EP2601216A1 - Kovalente diabodies und deren verwendung - Google Patents

Kovalente diabodies und deren verwendung

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Publication number
EP2601216A1
EP2601216A1 EP11815113.3A EP11815113A EP2601216A1 EP 2601216 A1 EP2601216 A1 EP 2601216A1 EP 11815113 A EP11815113 A EP 11815113A EP 2601216 A1 EP2601216 A1 EP 2601216A1
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EP
European Patent Office
Prior art keywords
domain
binding
diabody
polypeptide
antigen
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EP11815113.3A
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English (en)
French (fr)
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EP2601216A4 (de
EP2601216B1 (de
Inventor
Leslie S. Johnson
Ling Huang
Godfrey Jonah Anderson Rainey
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Macrogenics Inc
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Macrogenics Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2851—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/626—Diabody or triabody
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction

Definitions

  • the present invention is directed to diabody molecules, otherwise referred to as "dual affinity retargeting reagents" ("DARTS"), and uses thereof in the treatment of a variety of diseases and disorders, including immunological disorders and cancers.
  • the diabody molecules of the invention comprise at least two polypeptide chains that associate to form at least two epitope binding sites, which may recognize the same or different epitopes. Additionally, the epitopes may be from the same or different molecules or located on the same or different cells.
  • the individual polypeptide chains of the diabody molecule may be covalently bound through non-peptide bond covalent bonds, such as, but not limited to, disulfide bonding of cysteine residues located within each polypeptide chain.
  • the diabody molecules of the present invention further comprise an Fc region, which allows antibody-like functionality to be engineered into the molecule.
  • VL and VH domains are located on separate polypeptide chains, i.e., the light chain and the heavy chain, respectively.
  • the scFv construct comprises a VL and VH domain of an antibody contained in a single polypeptide chain wherein the domains are separated by a flexible linker of sufficient length to allow self-assembly of the two domains into a functional epitope binding site.
  • a linker of insufficient length less than about 12 amino acid residues
  • two of the scFv constructs interact with each other to form a bivalent molecule, the VL of one chain associating with the VH of the other (reviewed in Marvin et al.
  • VL and VH domains of differing specificity are selected, not only a bivalent, but also a bispecific molecule may be constructed.
  • Bivalent diabodies have wide ranging applications including therapy and immunodiagnosis. Bivalency allows for great flexibility in the design and engineering of diabody in various applications, providing enhanced avidity to multimeric antigens, the cross-linking of differing antigens, and directed targeting to specific cell types relying on the presence of both target antigens. Due to their increased valency, low dissociation rates and rapid clearance from the circulation (for diabodies of small size, at or below -50 kDa), diabody molecules known in the art have also shown particular use in the filed of tumor imaging (Fitzgerald et al. (1997) "Improved Tumour Targeting By Disulphide Stabilized Diabodies Expressed In Pichia pas tor is, " Protein Eng. 10: 1221). Of particular
  • Diabody epitope binding domains may also be directed to a surface determinant of any immune effector cell such as CD3, CD16, CD32, or CD64, which are expressed on T lymphocytes, natural killer (NK) cells or other mononuclear cells.
  • NK natural killer
  • diabody binding to effector cell determinants e.g. , Fey receptors (FcyR) was also found to activate the effector cell (Holliger et al. (1996) "Specific Killing Of Lymphoma Cells By Cytotoxic T-Cells Mediated By A Bispecific Diabody, " Protein Eng.
  • diabody molecules of the invention may exhibit Ig-like functionality independent of whether they comprise an Fc domain (e.g., as assayed in any efferctor function assay known in the art or exemplified herein (e.g. , ADCC assay)).
  • the diabody By cross-linking tumor and effector cells, the diabody not only brings the effector cell within the proximity of the tumor cells but leads to effective tumor killing (see e.g., Cao et al. (2003) "Bispecific Antibody Conjugates In
  • the Fey receptors members of the immunoglobulin gene superfamily of proteins, are surface glycoproteins that can bind the Fey portion of immunoglobulin molecules. Each member of the family recognizes immunoglobulins of one or more isotypes through a recognition domain on the alpha chain of the Fey receptor. Fey receptors are defined by their specificity for immunoglobulin subtypes. Fey receptors for IgG are referred to as FcyR, for IgE as FcsR, and for IgA as FcaR.
  • Different accessory cells bear Fey receptors for antibodies of different isotype, and the isotype of the antibody determines which accessory cells will be engaged in a given response (reviewed by Ravetch J.V. et al. (1991) "Fc Receptors," Annu. Rev. Immunol. 9: 457-92; Gerber J.S. et
  • Fey Receptors Rubor Redux, " Cell, 78(4): 553-60).
  • Table 1 adapted from Immunobiologv: The Immune System in Health and Disease, 4 th ed. 1999, Elsevier Science Ltd/Garland Publishing, New York).
  • Each member of this family is an integral membrane glycoprotein, possessing extracellular domains related to a C2-set of immunoglobulin-related domains, a single membrane spanning domain and an intracytoplasmic domain of variable length.
  • FcyRs designated FcyRI(CD64), FcyRII(CD32), and
  • FcyRIII(CD16) The three receptors are encoded by distinct genes; however, the extensive homology between the three family members suggest they arose from a common progenitor perhaps by gene duplication.
  • FcyRII proteins are 40 kDa integral membrane glycoproteins which bind only the complexed IgG due to a low affinity for monomeric Ig (10 6 M "1 ). This receptor is the most widely expressed FcyR, present on all hematopoietic cells, including monocytes, macrophages, B cells, NK cells, neutrophils, mast cells, and platelets. FcyRII has only two immunoglobulin-like regions in its immunoglobulin binding chain and hence a much lower affinity for IgG than FcyRI. There are three human FcyRII genes (FcyRII-A, FcyRII-B, FcyRII-C), all of which bind IgG in aggregates or immune complexes.
  • FcyRII-B create two functionally heterogenous responses to receptor ligation.
  • the fundamental difference is that the A isoform initiates intracellular signaling leading to cell activation such as phagocytosis and respiratory burst, whereas the B isoform initiates inhibitory signals, e.g. , inhibiting B-cell activation.
  • FcyRIII Due to heterogeneity within this class, the size of FcyRIII ranges between
  • FcyRIIIA an integral membrane glycoprotein
  • FcyRIIIB a glycosylphosphatidyl-inositol (GPI)- linked version.
  • FcyRIIIA an integral membrane glycoprotein
  • FcyRIIIB a glycosylphosphatidyl-inositol (GPI)- linked version.
  • GPI glycosylphosphatidyl-inositol
  • One murine gene encodes an FcyRIII homologous to the membrane spanning human FcyRIIIA.
  • FcyRIII shares structural characteristics with each of the other two FcyRs.
  • FcyRIIl binds IgG with low affinity and contains the corresponding two extracellular Ig-like domains.
  • FcyRIIIA is expressed in macrophages, mast cells and is the lone FcyR in NK cells.
  • the GPI-linked FcyRIIIB is currently known to be expressed only in human neutrophils.
  • Both activating and inhibitory signals are transduced through the FcyRs following ligation. These diametrically opposing functions result from structural differences among the different receptor isoforms.
  • Two distinct domains within the cytoplasmic signaling domains of the receptor called immunoreceptor tyrosine based activation motifs (ITAMs) or immunoreceptor tyrosine based inhibitory motifs (ITIMS) account for the different responses.
  • ITAMs immunoreceptor tyrosine based activation motifs
  • ITIMS immunoreceptor tyrosine based inhibitory motifs
  • FcyRIIA gene Human neutrophils express the FcyRIIA gene.
  • FcyRIIA clustering via immune complexes or specific antibody cross-linking serves to aggregate ITAMs along with receptor-associated kinases which facilitate ITAM phosphorylation.
  • ITAM phosphorylation serves as a docking site for Syk kinase, activation of which results in activation of downstream substrates ⁇ e.g., PI 3 K).
  • Cellular activation leads to release of proinflammatory mediators.
  • the FcyRIIB gene is expressed on B lymphocytes; its extracellular domain is 96% identical to FcyRIIA and binds IgG complexes in an indistinguishable manner.
  • the presence of an ITIM in the cytoplasmic domain of FcyRIIB defines this inhibitory subclass of FcyR. Recently the molecular basis of this inhibition was established.
  • the ITIM in FcyRIIB When co-ligated along with an activating FcyR, the ITIM in FcyRIIB becomes phosphorylated and attracts the SH2 domain of the inosital polyphosphate 5 ' -phosphatase (SHIP), which hydrolyzes phosphoinositol messengers released as a consequence of ITAM-containing FcyR- mediated tyrosine kinase activation, consequently preventing the influx of intracellular Ca ++ .
  • SHIP inosital polyphosphate 5 ' -phosphatase
  • crosslinking of FcyRIIB dampens the activating response to FcyR ligation and inhibits cellular responsiveness. B cell activation, B cell proliferation and antibody secretion is thus aborted.
  • the present invention relates to covalent diabodies and/or covalent diabody molecules and to their use in the treatment of a variety of diseases and disorders including cancer, autoimmune disorders, allergy disorders and infectious diseases caused by bacteria, fungi or viruses.
  • the diabody of the present invention can bind to two different epitopes on two different cells wherein the first epitope is expressed on a different cell type than the second epitope, such that the diabody can bring the two cells together.
  • the present invention is directed to a covalent bispecific diabody, which diabody comprises a first and a second polypeptide chain, which first polypeptide chain comprises (i) a first domain comprising a binding region of a light chain variable domain of a first immunoglobulin (VL1 ) specific for a first epitope, (ii) a second domain comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2) specific for a second epitope, and, optionally, (iii) a third domain comprising at least one cysteine residue, which first and second domains are covalently linked such that the first and second domains do not associate to form an epitope binding site; which second polypeptide chain comprises (i) a fourth domain comprising a binding region of a light chain variable domain of the second immunoglobulin (VL2), (ii) a fifth domain comprising a binding region of a heavy chain variable domain of the first immunoglobulin
  • the present invention is directed to a covalent bispecific diabody, which diabody comprises a first and a second polypeptide chain, which first polypeptide chain comprises (i) a first domain comprising a binding region of a light chain variable domain of a first immunoglobulin (VL1 ) specific for a first epitope, (ii) a second domain comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2) specific for a second epitope and (iii) a third domain comprising an Fc domain or portion thereof, which first and second domains are covalently linked such that the first and second domains do not associate to form an epitope binding site; which second polypeptide chain comprises (i) a fourth domain comprising a binding region of a light chain variable domain of the second immunoglobulin (VL2), (ii) a fifth domain comprising a binding region of a heavy chain variable domain of the first immunoglobulin (VHl),
  • the present invention is directed to diabody molecule, which molecule comprises a first and a second polypeptide chain, which first polypeptide chain comprises (i) a first domain comprising a binding region of a light chain variable domain of a first immunoglobulin (VL 1 ) specific for a first epitope, (ii) a second domain comprising a binding region of a heavy chain variable domain of a second
  • the present invention is directed to a covalent bispecific diabody, which diabody is a dimer of diabody molecules, each diabody molecule comprising a first and a second polypeptide chain, which first polypeptide chain comprises (i) a first domain comprising a binding region of a light chain variable domain of a first immunoglobulin (VL 1 ) specific for a first epitope, (ii) a second domain comprising a binding region of a heavy chain variable domain of a second
  • the present invention is directed to a covalent tetrapecific diabody, which diabody is a dimer of diabody molecules, the first diabody molecule comprising a first and a second polypeptide chain, which first polypeptide chain comprises (i) a first domain comprising a binding region of a light chain variable domain of a first immunoglobulin (VL1 ) specific for a first epitope, (ii) a second domain comprising a binding region of a heavy chain variable domain of a second
  • the first epitope, second epitope, and where applicable, third epitope and fourth epitope can be the same. In other aspects, the first epitope, second epitope, and where applicable, third epitope and fourth epitope can each different from the other. In certain aspects of the invention comprising a third epitope binding domain, the first epitope and third epitope can be the same. In certain aspects of the invention comprising a fourth epitope binding domain, the first epitope and fourth epitope can be the same. In certain aspects of the invention comprising a third epitope binding domain, the second epitope and third epitope can be the same.
  • the second epitope and fourth epitope can be the same.
  • the first eptitope and second epitope are different.
  • the third epitope and fourth epitope can be different. It is to be understood that any combination of the foregoing is encompassed in the present invention.
  • the first domain and the fifth domain of the diabody or diabody molecule can be derived from the same immunoglobulin.
  • the second domain and the fourth domain of the diabody or diabody molecule can be derived from the same immunoglobulin.
  • the first domain and the fifth domain of the diabody or diabody molecule can be derived from a different immunoglobulin.
  • the second domain and the fourth domain of the diabody or diabody molecule can be derived from a different
  • the covalent linkage between the first polypeptide chain and second polypeptide chain of the diabody or diabody molecule can be via a disulfide bond between at least one cysteine residue on the first polypeptide chain and at least one cysteine residue on the second polypeptide chain.
  • the cysteine residues on the first or second polypeptide chains that are responsible for disulfide bonding can be found anywhere on the polypeptide chain including within the first, second, third, fourth, fifth and sixth domains.
  • the cysteine residue on the first polypeptide chain is found in the first domain and the cysteine residue on the second polypeptide chain is found in the fifth domain.
  • the first, second, fourth and fifth domains correspond to the variable regions responsible for binding.
  • the cysteine residues responsible for the disulfide bonding between the first and second polypeptide chains are located within the third and sixth domains, respectively.
  • the third domain of the first polypeptide chain comprises the C-terminal 6 amino acids of the human kappa light chain, FNRGEC (SEQ ID NO: 23), which can be encoded by the amino acid sequence (SEQ ID NO: 17).
  • the sixth domain of the second polypeptide chain comprises the C-terminal 6 amino acids of the human kappa light chain, FNRGEC (SEQ ID NO: 23), which can be encoded by the amino acid sequence (SEQ ID NO: 17).
  • the third domain of the first polypeptide chain comprises the amino acid sequence VEP SC (SEQ ID NO: 77), derived from the hinge domain of a human IgG, and which can be encoded by the nucleotide sequence (SEQ ID NO: 78).
  • the sixth domain of the second polypeptide chain comprises the C-terminal 6 amino acids of the human kappa light chain, FNRGEC (SEQ ID NO: 23), which can be encoded by the amino acid sequence (SEQ ID NO: 17).
  • the third domain of the first polypeptide chain comprises the amino acid sequence VEP SC (SEQ ID NO: 77), derived from the hinge domain of a human IgG, and which can be encoded by the nucleotide sequence (SEQ ID NO: 78).
  • polypeptide chain comprises the amino acid sequence VEPKSC (SEQ ID NO: 77), derived from the hinge domain of a human IgG, and which can be encoded by the nucleotide sequence (SEQ ID NO: 78).
  • the third domain of the first polypeptide chain comprises the C-terminal 6 amino acids of the human kappa light chain, FNRGEC (SEQ ID NO: 23); and the sixth domain of the second polypeptide chain comprises the amino acid sequence VEPKSC (SEQ ID NO: 77).
  • the sixth domain of the second polypeptide chain comprises the C-terminal 6 amino acids of the human kappa light chain, FNRGEC (SEQ ID NO: 23); and the third domain of the first polypeptide chain comprises the amino acid sequence VEPKSC (SEQ ID NO: 77).
  • the third domain of the first polypeptide chain comprises the C-terminal 6 amino acids of the human kappa light chain, FNRGEC (SEQ ID NO: 23); and the sixth domain of the second polypeptide chain comprises a hinge domain.
  • the sixth domain of the second polypeptide chain comprises the C-terminal 6 amino acids of the human kappa light chain, FNRGEC (SEQ ID NO: 23); and the third domain of the first polypeptide chain comprises the hinge domain.
  • the third domain of the first polypeptide chain comprises the C-terminal 6 amino acids of the human kappa light chain, FNRGEC (SEQ ID NO: 23); and the sixth domain of the first polypeptide chain comprises an Fc domain, or portion thereof.
  • the sixth domain of the second polypeptide chain comprises the C-terminal 6 amino acids of the human kappa light chain, FNRGEC (SEQ ID NO: 23); and the third domain of the first polypeptide chain comprises an Fc domain, or portion thereof.
  • the cysteine residues on the first or second polypeptide that are responsible for the disulfide bonding can be located outside of the first, second or third domains on the first polypeptide chain and outside of the fourth, fifth and sixth domain on the second polypeptide chain.
  • the cysteine residue on the first polypeptide chain can be N-terminal to the first domain or can be C-terminal to the first domain.
  • the cysteine residue on the first polypeptide chain can be N-terminal to the second domain or can be C-terminal to the second domain.
  • the cysteine residue on the first polypeptide chain can be N-terminal to the third domain or can be C-terminal to the third domain.
  • cysteine residue on the second polypeptide chain can be N- terminal to the fourth domain or can be C-terminal to the fourth domain.
  • the cysteine residue on the second polypeptide chain can be N-terminal to the fifth domain or can be C- terminal to the fifth domain.
  • the cysteine residue on the second polypeptide chain can be C-terminal to the sixth domain or can be N-terminal to the sixth domain.
  • disulfide bond can between at least two cysteine residues on the first polypeptide chain and at least two cysteine residues on the second polypeptide chain.
  • the cysteine residue can be at the C-terminus of the first polypeptide chain and at the C-terminus of the second polypeptide chain. It is to be understood that any combination of the foregoing is encompassed in the present invention.
  • the covalent diabody of the invention encompasses dimers of diabody molecules, wherein each diabody molecule comprises a first and second polypeptide chain.
  • the diabody molecules can be covalently linked to form the dimer, with the proviso that the covalent linkage is not a peptide bond.
  • the covalent linkage is a disulfide bond between at least one cysteine residue on the first polypeptide chain of each of the diabody molecules of the dimer.
  • the covalent linkage is a disulfide bond between at least one cysteine residue on the first polypeptide chain of each of the diabody molecules forming the dimer, wherein said at least one cysteine residue is located in the third domain of each first polypeptide chain.
  • the first domain on the first polypeptide chain can be N-terminal to the second domain or can be C-terminal to the second domain.
  • the first domain on the first polypeptide chain can be N-terminal to the third domain or can be C-terminal to the third domain.
  • the second domain on the first polypeptide chain can be N-terminal to the first domain or can be C-terminal to the first domain.
  • the second domain on the first polypeptide chain can be N-terminal to the third domain or can be C-terminal to the third domain.
  • the third domain on the first polypeptide chain can be N-terminal to the first domain or can be C-terminal to the first domain.
  • the third domain on the first polypeptide chain can be N-terminal to the second domain or can be C-terminal to the second domain.
  • the fourth domain can be N-terminal to the fifth domain or can be C-terminal to the fifth domain.
  • the fourth domain can be N-terminal to the sixth domain or can be C-terminal to the sixth domain.
  • the fifth domain on the second polypeptide chain can be N-terminal to the fourth domain or can be C-terminal to the fourth domain.
  • the fifth domain on the second polypeptide chain can be N-terminal to the sixth domain or can be C-terminal to the sixth domain.
  • the sixth domain on the second polypeptide chain can be N-terminal to the fourth domain or can be C-terminal to the fourth domain.
  • the sixth domain on the second polypeptide chain can be N-terminal to the fifth domain or can be C-terminal to the fifth domain. It is to be understood that any combination of the foregoing is encompassed in the present invention.
  • first domain and second domain can be located C- terminal to the third domain on the first polypeptide chain; or the first domain and second domain can be located N-terminal to the third domain on the first polypeptide chain.
  • the fourth domain and fifth domain can be located C-terminal to the sixth domain, or the fourth domain and fifth domain can be located N- terminal to the sixth domain.
  • the present invention is directed to a covalent bispecific diabody, which diabody is a dimer of diabody molecules, each diabody molecule comprising a first and a second polypeptide chain, which first polypeptide chain comprises (i) a first domain comprising a binding region of a light chain variable domain of a first immunoglobulin (VL1 ) specific for a first epitope, (ii) a second domain comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2) specific for a second epitope and (iii) a third domain comprising an Fc domain or portion thereof, which first and second domains are covalently linked such that the first and second domains do not associate to form an epitope binding site and wherein the third domain is located N-terminal to both the first domain and second domain; and which second polypeptide chain comprises (i) a fourth domain comprising a binding region of a light chain variable domain of the second immunoglobulin
  • VL2(VH2) (VL2)(VH2) that binds the second epitope.
  • the present invention is directed to a covalent tetrapecific diabody, which diabody is a dimer of diabody molecules, the first diabody molecule comprising a first and a second polypeptide chain, which first polypeptide chain comprises (i) a first domain comprising a binding region of a light chain variable domain of a first immunoglobulin (VL1 ) specific for a first epitope, (ii) a second domain comprising a binding region of a heavy chain variable domain of a second
  • the domains on the individual polypeptide chains are covalently linked.
  • the covalent link between the first and second domain, first and third domain, second and third domain, fourth and fifth domain, fourth and sixth domain, and/or fifth and sixth domain can be a peptide bond.
  • the first and second domains, and the fourth and fifth domains can be separated by the third domain and sixth domain, respectively, or by additional amino acid residues, so long as the first and second, and fourth and fifth domains do not associate to form a binding site.
  • the number of amino acid residues can be 0, 1 , 2, 3, 4, 5, 6, 7, 8 or 9 amino acid residues. In one preferred aspect, the number of amino acid residues between the domains is 8.
  • the domains of the first and second polypeptid chain comprising an Fc domain can further comprise a hinge domain such that the domain comprises a hinge- Fc region.
  • the first polypeptide chain or the second polypeptide chain can comprise a hinge domain without also comprising an Fc domain.
  • the heavy chains, light chains, hinge regions, Fc domains, and/or hinge-Fc domains for use in the invention can be derived from any immunoglobulin type including IgA, IgD, IgE, IgG or IgM.
  • the immunoglobulin type is IgG, or any subtype thereof, i.e. , IgGi, IgG 2 , IgG 3 or IgG 4 .
  • the immunoglobulin from which the light and heavy chains are derived is humanized or chimerized.
  • the first epitope and second epitopes, and, where applicable, third epitope and fourth epitope, to which the diabody or diabody molecule binds can be different epitopes from the same antigen or can be different epitopes from different antigens.
  • the antigens can be any molecule to which an antibody can be generated. For example, proteins, nucleic acids, bacterial toxins, cell surface markers, autoimmune markers, viral proteins, drugs, etc.
  • at least one epitope binding site of the diabody is specific for an antigen on a particular cell, such as a B-cell, a T-cell, a phagocytic cell, a natural killer (N ) cell or a dendritic cell.
  • At least one epitope binding site of the diabody or diabody molecule is specific for a Fc receptor, which Fc receptor can be an activating Fc receptor or an inhibitory Fc receptor.
  • the Fc receptor is a Fey receptor
  • the Fey receptor is a FcyRI, FcyRII or FcyRIII receptor.
  • the FcyRIII receptor is the FcyRIIIA (CD16A) receptor or the FcyRIIIB (CD16B) receptor, and, more preferably, the FcyRIII receptor is the FcyRIIIA (CD16A) receptor.
  • the FcyRII receptor is the FcyRIIA (CD32A) receptor or the FcyRIIB (CD32B) receptor, and more preferably the FcyRIIB (CD32B) receptor.
  • one binding site of the diabody is specific for CD32B and the other binding site is specific for CD16A.
  • at least one epitope binding site of the diabody or diabody molecule is specific for an activating Fc receptor and at least one other site is specific for an inhibitory Fc receptor.
  • the activating Fc receptor is CD32A and the inhibitory Fc receptor is CD32B.
  • the activating Fc receptor is BCR and the inhibitory Fc receptor is CD32B.
  • the activating Fc receptor is IgERI and the inhibitory Fc receptor is CD32B.
  • VH domains can be the same as or similar to the VL and VH domains of the mouse antibody 3G8, the sequence of which has been cloned and is set forth herein.
  • the VL and VH domains can be the same as or similar to the VL and VH domains of the mouse antibody IV.3.
  • the VL and VH domains can be the same as or similar to the VL and VH domains of the mouse antibody 2B6, the sequence of which has been cloned and is set forth herein.
  • VL or VH domains of the 3G8, 2B6 and IV.3 antibodies can be used in any combination.
  • the present invention is also directed to a bispecific diabody or diabody molecule wherein the first epitope is specific for CD32B, and the second epitope is specific for CD16A.
  • an epitope binding site can be specific for a pathogenic antigen.
  • a pathogenic antigen is an antigen involved in a specific pathogenic disease, including cancer, infection and autoimmune disease.
  • the pathogenic antigen can be a tumor antigen, a bacterial antigen, a viral antigen, or an autoimmune antigen.
  • pathogenic antigens include, but are not limited to lipopolysaccharide, viral antigens selected from the group consisting of viral antigens from human immunodeficiency virus, Adenovirus, Respiratory Syncitial Virus, West Nile Virus (e.g., E16 and/or E53 antigens) and hepatitis virus, nucleic acids (DNA and RNA) and collagen.
  • the pathogenic antigen is a neutralizing antigen.
  • the other epitope binding site is specific for a pathogenic antigen excluding autoimmune antigens.
  • the other epitope binding site is specific for any pathogenic antigen.
  • the diabody molecule of the invention binds two different antigens on the same cell, for example, one antigen binding site is specific for an activating Fc receptor while the other is specific for an inhibitory Fc receptor.
  • the diabody molecule binds two distinct viral neutralizing epitopes, for example, but not limited to, El 6 and E53 of West Nile Virus.
  • the diabodies of the invention can be used to treat a variety of diseases and disorders. Accordingly, the present invention is directed to a method for treating a disease or disorder comprising
  • a covalent diabody or diabody molecule of the invention in which at least one binding site is specific for a pathogenic antigen, such as an antigen expressed on the surface of a cancer cell or on the surface of a bacterium or virion and at least one other binding site is specific for a Fc receptor, e.g. , CD16A.
  • a pathogenic antigen such as an antigen expressed on the surface of a cancer cell or on the surface of a bacterium or virion
  • at least one other binding site is specific for a Fc receptor, e.g. , CD16A.
  • the invention is directed to a method for treating a disease or disorder comprising administering to a patient in need thereof an effective amount of a diabody or diabody molecule of the invention, in which at least one binding site is specific for CD32B and at least one other binding site is specific for CD16A.
  • the invention is directed to a method for inducing immune tolerance to a pathogenic antigen comprising administering to a patient in need there an effective amount of a covalent diabody or dovalent diabody molecule of the invention, in which at least one binding site is specific for CD32B and at least one other binding site is specific for said pathogenic antigen.
  • the pathogenic antigen can be an allergen or another molecule to which immune tolerance is desired, such as a protein expressed on transplanted tissue.
  • the present invention is directed to a method for detoxification comprising administering to a patient in need thereof an effective amount of a covalent diabody or diabody molecule of the invention, in which at least one binding site is specific for a cell surface marker and at least one other binding site is specific for a toxin.
  • the diabody of the invention administered is one where one binding site is specific for a cell surface marker such as an Fc and the other binding site is specific for a bacterial toxin or for a drug.
  • the cell surface marker is not found on red blood cells.
  • antibody refers to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, polyclonal antibodies, camelized antibodies, single-chain Fvs (scFv), single chain antibodies, Fab fragments, F(ab " ) fragments, disulfide-linked bispecific Fvs (sdFv), intrabodies, and anti-idiotypic (anti-Id) antibodies (including, e.g. , anti-Id and anti-anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above.
  • scFv single-chain Fvs
  • Fab fragments fragments
  • F(ab " ) fragments fragments
  • disulfide-linked bispecific Fvs sdFv
  • intrabodies and anti-idiotypic (anti-Id) antibodies (including, e.g. , anti-Id and anti-anti-Id antibodies to antibodies of the invention), and epitope-binding fragment
  • antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site.
  • Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g. , IgG,, IgG 2 , IgG 3 , IgG 4 , IgA t and IgA 2 ) or subclass.
  • immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g. , IgG,, IgG 2 , IgG 3 , IgG 4 , IgA t and IgA 2 ) or subclass.
  • immunospecifically recognizes refers to molecules that specifically bind to an antigen (e.g., eptiope or immune complex) and do not specifically bind to another molecule.
  • a molecule that specifically binds to an antigen may bind to other peptides or polypeptides with lower affinity as determined by, e.g., immunoassays, BIAcore, or other assays known in the art.
  • molecules that specifically bind an antigen do not cross-react with other proteins.
  • Molecules that specifically bind an antigen can be identified, for example, by immunoassays, BIAcore, or other techniques known to those of skill in the art.
  • immune complex refers to a structure which forms when at least one target molecule and at least one heterologous Fey region-containing polypeptide bind to one another forming a larger molecular weight complex.
  • immune complexes are antigen-antibody complexes which can be either soluble or particulate (e.g., an antigen/antibody complex on a cell surface.).
  • frame region has their ordinary meaning in the immunology art and refer to domains in naturally occurring immunoglobulins and the corresponding domains of synthetic (e.g., recombinant) binding proteins (e.g. , humanized antibodies, single chain antibodies, chimeric antibodies, etc.).
  • the basic structural unit of naturally occurring immunoglobulins e.g., IgG
  • the amino- terminal (“N") portion of each chain includes a variable region of about 100 to 1 10 or more amino acids primarily responsible for antigen recognition.
  • the carboxy-terminal (“C”) portion of each chain defines a constant region, with light chains having a single constant domain and heavy chains usually having three constant domains and a hinge region.
  • the structure of the light chains of an IgG molecule is ⁇ -VL-CL-C and the structure of IgG heavy chains is n-VH-CHi -H-Qa-Qu-c (where H is the hinge region).
  • the variable regions of an IgG molecule consist of the complementarity determining regions (CDRs), which contain the residues in contact with antigen and non-CDR segments, referred to as framework segments, which in general maintain the structure and determine the positioning of the CDR loops (although certain framework residues may also contact antigen).
  • the V L and VH domains have the structure n-FRl , CDR1 , FR2, CDR2, FR3, CDR3, FR4-c. .
  • the assignment of amino acids to each domain is in accordance with the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991). Amino acids from the variable regions of the mature heavy and light chains of immunoglobulins are designated by the position of an amino acid in the chain. Kabat described numerous amino acid sequences for antibodies, identified an amino acid consensus sequence for each subgroup, and assigned a residue number to each amino acid.
  • Kabat' s numbering scheme is extendible to antibodies not included in his compendium by aligning the antibody in question with one of the consensus sequences in Kabat by reference to conserved amino acids.
  • This method for assigning residue numbers has become standard in the field and readily identifies amino acids at equivalent positions in different antibodies, including chimeric or humanized variants. For example, an amino acid at position 50 of a human antibody light chain occupies the equivalent position to an amino acid at position 50 of a mouse antibody light chain.
  • the term "heavy chain” is used to define the heavy chain of an IgG antibody.
  • the heavy chain comprises the immunoglobulin domains VH, CHI , hinge, CH2 and CH3.
  • the numbering of the residues in an IgG heavy chain is that of the EU index as in Kabat et al, Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service, NHl , MD (1991 ), expressly incorporated herein by references.
  • the "EU index as in Kabat” refers to the numbering of the human IgGl EU antibody. Examples of the amino acid sequences containing human IgGl hinge, CH2 and CH3 domains are shown in FIGS.
  • FIGS. 1A and IB also set forth amino acid sequences of the hinge, CH2 and CH3 domains of the heavy chains of IgG2, IgG3 and IgG4.
  • the amino acid sequences of IgG2, IgG3 and IgG4 isotypes are aligned with the IgGl sequence by placing the first and last cysteine residues of the respective hinge regions, which form the inter- heavy chain S-S bonds, in the same positions.
  • the EU index For the IgG2 and IgG3 hinge region, not all residues are numbered by the EU index.
  • the "hinge region” or “hinge domain” is generally defined as stretching from Glu216 to Pro230 of human IgGl .
  • An example of the amino acid sequence of the human IgG l hinge region is shown in FIG. 1A (amino acid residues in FIG. 1A are numbered according to the Kabat system). Hinge regions of other IgG isotypes may be aligned with the IgGl sequence by placing the first and last cysteine residues forming inter-heavy chain S-S binds in the same positions as shown in FIG. 1A.
  • Fc region As used herein, the term "Fc region,” “Fc domain” or analogous terms are used to define a C-terminal region of an IgG heavy chain.
  • An example of the amino acid sequence containing the human IgGl is shown in FIG. IB. Although boundaries may vary slightly, as numbered according to the Kabat system, the Fc domain extends from amino acid 231 to amino acid 447 (amino acid residues in FIG. IB are numbered according to the Kabat system).
  • FIG. IB also provides examples of the amino acid sequences of the Fc regions of IgG isotypes IgG2, IgG3, and IgG4.
  • the Fc region of an IgG comprises two constant domains, CH2 and CH3.
  • the CH2 domain of a human IgG Fc region usually extends from amino acids 231 to amino acid 341 according to the numbering system of Kabat (FIG. I B).
  • the CH3 domain of a human IgG Fc region usually extends from amino acids 342 to 447 according to the numbering system of Kabat (FIG. I B).
  • the CH2 domain of a human IgG Fc region (also referred to as "Cy2" domain) is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG.
  • FcyR binding protein As used herein the terms "FcyR binding protein,” “FcyR antibody,” and
  • anti-FcyR antibody are used interchangeably and refer to a variety of immunoglobulin- like or immunoglobulin-derived proteins.
  • FcyR binding proteins bind FcyR via an interaction with VL and/or V H domains (as distinct from Fcy-mediated binding).
  • Examples of FcyR binding proteins include fully human, polyclonal, chimeric and humanized antibodies (e.g. , comprising 2 heavy and 2 light chains), fragments thereof (e.g. , Fab, Fab', F(ab') 2 , and Fv fragments), Afunctional or multifunctional antibodies (see, e.g. ,
  • the FcyRIIIA binding protein is a "tetrameric antibody" i.e., having generally the structure of a naturally occurring IgG and comprising variable and constant domains, i.e. , two light chains comprising a VL domain and a light chain constant domain and two heavy chains comprising a V H domain and a heavy chain hinge and constant domains.
  • FcyR antagonists and analogous terms refer to protein and non-proteinacious substances, including small molecules which antagonize at least one biological activity of an VcyR, e.g., block signaling.
  • the molecules of the invention block signaling by blocking the binding of IgGs to an FcyR.
  • derivative in the context of polypeptides or proteins refers to a polypeptide or protein that comprises an amino acid sequence which has been altered by the introduction of amino acid residue substitutions, deletions or additions.
  • derivative as used herein also refers to a polypeptide or protein which has been modified, i.e, by the covalent attachment of any type of molecule to the polypeptide or protein.
  • an antibody may be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular an antigen or other protein, etc.
  • a derivative polypeptide or protein may be produced by chemical modifications using techniques known to those of skill in the art, including, but not limited to specific chemical cleavage, acetylation. formylation, metabolic synthesis of tunicamycin, etc. Further, a derivative polypeptide or protein derivative possesses a similar or identical function as the polypeptide or protein from which it was derived.
  • the term "derivative" in the context of a non-proteinaceous derivative refers to a second organic or inorganic molecule that is formed based upon the structure of a first organic or inorganic molecule.
  • a derivative of an organic molecule includes, but is not limited to, a molecule modified, e.g. , by the addition or deletion of a hydroxyl, methyl, ethyl, carboxyl or amine group.
  • An organic molecule may also be esterified, alkylated and/or phosphorylated.
  • the term "diabody molecule” refers to a complex of two or more polypeptide chains or proteins, each comprising at least one VL and one VH domain or fragment thereof, wherein both domains are comprised within a single polypeptide chain.
  • "diabody molecule” includes molecules comprising an Fc or a hinge-Fc domain. Said polypeptide chains in the complex may be the same or different, i.e., the diabody molecule may be a homo-multimer or a hetero-multimer.
  • “diabody molecule” includes dimers or tetramers or said polypeptide chains containing both a VL and VH domain. The individual polypeptide chains comprising the multimeric proteins may be covalently joined to at least one other peptide of the multimer by interchain disulfide bonds.
  • autoimmune disease is used interchangeably with the term “autoimmune disorder” to refer to a condition in a subject characterized by cellular, tissue and/or organ injury caused by an immunologic reaction of the subject to its own cells, tissues and/or organs.
  • inflammatory disease is used interchangeably with the term “inflammatory disorder” to refer to a condition in a subject characterized by inflammation, preferably chronic inflammation.
  • Autoimmune disorders may or may not be associated with inflammation.
  • inflammation may or may not be caused by an autoimmune disorder.
  • certain disorders may be characterized as both autoimmune and inflammatory disorders.
  • identical polypeptide chains as used herein also refers to polypeptide chains having almost identical amino acid sequence, for example, including chains having one or more amino acid differences, preferably conservative amino acid substitutions, such that the activity of the two polypeptide chains is not significantly different
  • cancer refers to a neoplasm or tumor resulting from abnormal uncontrolled growth of cells.
  • cancer explicitly includes, leukemias and lymphomas.
  • cancer refers to a benign tumor, which has remained localized.
  • cancer refers to a malignant tumor, which has invaded and destroyed neighboring body structures and spread to distant sites.
  • the cancer is associated with a specific cancer antigen.
  • an immunomodulatory agent refers to an agent that modulates a host's immune system.
  • an immunomodulatory agent is an immunosuppressant agent.
  • an immunomodulatory agent is an immunostimulatory agent.
  • Immunomodatory agents include, but are not limited to, small molecules, peptides, polypeptides, fusion proteins, antibodies, inorganic molecules, mimetic agents, and organic molecules.
  • epitope refers to a fragment of a polypeptide or protein or a non-protein molecule having antigenic or immunogenic activity in an animal, preferably in a mammal, and most preferably in a human.
  • immunogenic activity is a fragment of a polypeptide or protein that elicits an antibody response in an animal.
  • An epitope having antigenic activity is a fragment of a polypeptide or protein to which an antibody immunospecifically binds as determined by any method well-known to one of skill in the art, for example by immunoassays.
  • Antigenic epitopes need not necessarily be immunogenic.
  • fragment refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least contiguous 80 amino acid residues, at least contiguous 90 amino acid residues, at least contiguous 100 amino acid residues, at least contiguous 125 amino acid residues, at least 150 contiguous amino acid residues, at least contiguous 175 amino acid residues, at least contiguous 200 amino acid residues, or at least contiguous 250 amino acid residues of the amino acid sequence of another polypeptide.
  • a fragment of a polypeptide retains at least one function of
  • nucleic acids and “nucleotide sequences” include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), combinations of DNA and RNA molecules or hybrid DNA/RNA molecules, and analogs of DNA or RNA molecules.
  • Such analogs can be generated using, for example, nucleotide analogs, which include, but are not limited to, inosine or tritylated bases.
  • Such analogs can also comprise DNA or RNA molecules comprising modified backbones that lend beneficial attributes to the molecules such as, for example, nuclease resistance or an increased ability to cross cellular membranes.
  • the nucleic acids or nucleotide sequences can be single-stranded, double-stranded, may contain both single-stranded and double- stranded portions, and may contain triple-stranded portions, but preferably is
  • a "therapeutically effective amount” refers to that amount of the therapeutic agent sufficient to treat or manage a disease or disorder.
  • therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the onset of disease, e.g., delay or minimize the spread of cancer.
  • a therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease.
  • a therapeutically effective amount with respect to a therapeutic agent of the invention means the amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of a disease.
  • prophylactic agent and “prophylactic agents” refer to any agent(s) which can be used in the prevention of a disorder, or prevention of recurrence or spread of a disorder.
  • a prophylactically effective amount may refer to the amount of prophylactic agent sufficient to prevent the recurrence or spread of
  • hyperproliferative disease particularly cancer, or the occurrence of such in a patient, including but not limited to those predisposed to hyperproliferative disease, for example those genetically predisposed to cancer or previously exposed to carcinogens.
  • a prophylactically effective amount may also refer to the amount of the prophylactic agent that provides a prophylactic benefit in the prevention of disease. Further, a
  • prophylactically effective amount with respect to a prophylactic agent of the invention means that amount of prophylactic agent alone, or in combination with other agents, that provides a prophylactic benefit in the prevention of disease.
  • the terms “prevent”, “preventing” and “prevention” refer to the prevention of the recurrence or onset of one or more symptoms of a disorder in a subject as result of the administration of a prophylactic or therapeutic agent.
  • the term "in combination” refers to the use of more than one prophylactic and/or therapeutic agents.
  • the use of the term “in combination” does not restrict the order in which prophylactic and/or therapeutic agents are administered to a subject with a disorder.
  • a first prophylactic or therapeutic agent can be administered prior to (e.g., 5 minutes, 1 5 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g.
  • Appector function as used herein is meant a biochemical event that results from the interaction of an antibody Fc region with an Fc receptor or an antigen. Effector functions include but are not limited to antibody dependent cell mediated cytotoxicity (ADCC), antibody dependent cell mediated phagocytosis (ADCP), and complement dependent cytotoxicity (CDC). Effector functions include both those that operate after the binding of an antigen and those that operate independent of antigen binding.
  • ADCC antibody dependent cell mediated cytotoxicity
  • ADCP antibody dependent cell mediated phagocytosis
  • CDC complement dependent cytotoxicity
  • Effective cell as used herein is meant a cell of the immune system that expresses one or more Fc receptors and mediates one or more effector functions. Effector cells include but are not limited to monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans' cells, natural killer (NK) cells, and may be from any organism including but not limited to humans, mice, rats, rabbits, and monkeys.
  • Effector cells include but are not limited to monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans' cells, natural killer (NK) cells, and may be from any organism including but not limited to humans, mice, rats, rabbits, and monkeys.
  • the term "specifically binds an immune complex" and analogous terms refer to molecules that specifically bind to an immune complex and do not specifically bind to another molecule.
  • a molecule that specifically binds to an immune complex may bind to other peptides or polypeptides with lower affinity as determined by, e.g., immunoassays, BIAcore, or other assays known in the art.
  • molecules that specifically bind an immune complex do not cross-react with other proteins.
  • Molecules that specifically bind an immune complex can be identified, for example, by immunoassays, BIAcore, or other techniques known to those of skill in the art.
  • a "stable fusion protein” as used herein refers to a fusion protein that undergoes minimal to no detectable level of degradation during production and/or storage as assessed using common biochemical and functional assays known to one skilled in the art, and can be stored for an extended period of time with no loss in biological activity, e.g., binding to FcyR.
  • FIGS. 1 A-B AMINO ACID SEQUENCE OF HUMAN IgG CHI, HINGE and Fc
  • Figure 1 provides the amino acid sequences of human IgGl , IgG2, IgG3 and IgG4 hinge (A) and Fc (B) domains.
  • IgGl hinge domain SEQ ID NO: 1
  • IgG2 hinge domain SEQ ID NO: 2
  • IgG3 hinge domain SEQ ID NO: 3
  • IgG4 hinge domain SEQ ID NO: 4
  • IgGl Fc domain SEQ ID NO: 5
  • IgG2 Fc domain SEQ ID NO: 6
  • IgG3 Fc domain SEQ ID NO: 7
  • IgG l Fc domain SEQ ID NO: 8».
  • Isotype sequences are aligned with the IgG l sequence by placing the first and last cysteine residues of the respective hinge regions, which form the inter-heavy chain S- S bonds, in the same positions.
  • residues in the CH2 domain are indicated by +, while residues in the CH3 domain are indicated by ⁇ .
  • FIG. 2 SCHEMATIC REPRESENTATION OF POLYPEPTIDE CHAINS
  • Polypeptides of a covalent, bifunctional diabody consist of an antibody VL and an antibody VH domain separated by a short peptide linker.
  • the 8 amino acid residue linker prevents self assembly of a single polypeptide chain into scFv constructs, and, instead, interactions between the VL and VH domains of differing polypeptide chains predominate.
  • 4 constructs were created (each construct is described from the amino terminus ("n"), left side of the construct, to the carboxy terminus ("c"), right side of figure): construct (1) (SEQ ID NO: 9) comprised, n-the VL domain Hu2B6 - linker
  • FIG. 3 SDS-PAGE ANALYSIS OF AFFINITY PURIFIED DIABODIES
  • Affinity purified diabodies were subjected to SDS-PAGE analysis under reducing (lanes 1 -3) or non-reducing (lanes 4-6) conditions. Approximate molecular weights of the standard (in between lanes 3 and 4) are indicated. Lanes 1 and 4, h3G8 CMD; Lanes 2 and 5, h2B6 CMD; and Lanes 3 and 6, h2B6-h3G8 CBD.
  • FIGS. 4 A-B SEC ANALYSIS OF AFFINITY PURIFIED DIABODIES
  • Affinity purified diabodies were subjected to SEC analysis.
  • A Elution profile of known standards: full-length IgG (-150 kDa), Fab fragment of IgG (-50 kDa), and scFv (-30 kDa);
  • B Elution profile of h2b6 CMD, h3G8 CMD, and h2B6-h3G8 CBD.
  • FIG. 5 BINDING OF h2B6-h3G8 CBD TO sCD32B AND sCD16A
  • FIGS. 6 A C BIACORE ANALYSIS OF DIABODY BINDING TO sCD16A
  • FIGS. 7 A C BIACORE ANALYSIS OF DIABODY BINDING TO sCD16A and
  • h2B6-h3G8 CBD Binding of h2B6-h3G8 CBD to sCD32B.
  • Diabodies were injected at concentrations of 6.25-200 nM over receptor surfaces at a flow rate of 70 ml/min for 180 sec.
  • FIG. 8 SCHEMATIC DEPICTING THE INTERACTION OF
  • NH 2 and COOH represent the amino-terminus and carboxy terminus, respectively of each polypeptide chain.
  • S represents the C-terminal cysteine residue on each polypeptide chain.
  • VL and VH indicate the variable light domain and variable heavy domain, respectively. Dotted and dashed lines are to distinguish between the two polypeptide chains and, in particular, represent the linker portions of said chains.
  • h2B6 Fv and h3G8 Fv indicate an epitope binding site specific for CD32B and CD 16, respectively.
  • FIG. 9 SCHEMATIC REPRESENTATION OF POLYPEPTIDE CHAINS
  • GGGSGGGG SEQ ID NO: 10
  • LGGC second linker
  • FIG.10 BINDING OF DIABODY MOLECULES COMPRISING Fc
  • Diabodies assayed were produced by 3 recombinant expression systems: cotransfection of pMGX669 and pMGX674, expressing constructs 1 and 6, respectively; cotransfection of pMGX667 and pMGX676, expressing constructs 2 and 5, respectively; and cotransfection of pMGX674 and pMGX676, expressing constructs 5 and 6, respectively.
  • sCD32B was used as the target protein.
  • the secondary probe was HRP conjugated sCD16A.
  • FIG. 11 SCHEMATIC DEPICTING THE INTERACTION OF TWO
  • NH 2 and COOH represent the amino-terminus and carboxy terminus, respectively of each polypeptide chain.
  • S represents the at least one disulfide bond between a cysteine residue in the second linker sequence of each polypeptide chain.
  • VL and VH indicate the variable light domain and variable heavy domain, respectively.
  • CH2 and CH3 represent the
  • h2B6 Fv and h3G8 Fv indicate an epitope binding site specific for CD32B and CD 16, respectively.
  • FIG.12 BINDING OF DIABODY MOLECULES COMPRISING
  • Diabodies assayed were produced by 4 recombinant expression systems: cotransfection of pMGX669 + pMGX674, expressing constructs 1 and 6, respectively; cotransfection of pMGX669 + pMGX678, expressing constructs 2 and 8, respectively; cotransfection of pMGX677 + pMGX674, expressing constructs 7 and 6, respectively; and cotransfection of pMGX677 + pMGX678, expressing constructs 7 and 8, respectively.
  • sCD32B was used as the target protein.
  • the secondary probe was HRP conjugated sCD16A.
  • FIG. 13 SCHEMATIC DEPICTING THE INTERACTION OF
  • NH 2 and COOH represent the amino-terminus and carboxy terminus, respectively of each polypeptide chain.
  • S represents the at least one disulfide bond between a cysteine residue in the second linker sequence the Fc bearing, 'heavier,' polypeptide chain and a cysteine residue in the C-terminal sequence of the non-Fc bearing,
  • VL and VH indicate the variable light domain and variable heavy domain, respectively. Dotted and dashed lines are to distinguish between polypeptide chains and, in particular, represent the first linker portions of said heavier chains or the linker of said lighter chains.
  • CH2 and CH3 represent the CH2 and CH3 constant domains of an Fc domain.
  • h2B6 Fv and h3G8 Fv indicate an epitope binding site specific for CD32B and CD 16, respectively.
  • FIG. 14 SCHEMATIC REPRESENTATION OF POLYPEPTIDES
  • SEQ ID NO: 19 comprised n-a Hinge/Fc domain of human IgGl - the VL domain
  • Hu3G8 - linker (GGGSGGGG (SEQ ID NO: 10)) - the VH domain of Hu2B6 - linker
  • (SEQ ID NO: 20) comprised n-an Fc domain of human IgG l - the VL domain Hu3G8 - linker (GGGSGGGG (SEQ ID NO: 10)) - the VH domain of Hu2B6 - linker (GGGSGGGG (SEQ ID NO: 10))- and a C-terminal LGGC sequence-c; construct (1 1) (SEQ ID NO: 21) comprised n-the VL domain Hu2B6 (G105C) - linker (GGGSGGGG (SEQ ID NO: 10)) - the VH domain of Hu3G8 - and a C-terminal hinge/Fc domain of human IgGl with amino acid substitution A215V-c; construct (12) (SEQ ID NO: 22) comprised n-the VL domain Hu3G8 - linker (GGGSGGGG (SEQ ID NO: 10)) - the VH domain of Hu2B6 (G44C) - and a C-terminal FNRG
  • FIG. 15 A-B SDS-PAGE AND WESTERN BLOT ANALYSIS OF AFFINITY
  • Diabodies produced by recombinant expression systems cotransfected with vectors expressing constructs 10 and 1 , constructs 9 and 1 , and constructs 1 1 and 12 were subjected to SDS-PAGE analysis non-reducing conditions (A) and Western Blot analysis using goat anti-human IgGl H+L as the probe (B). Proteins in the SDS-PAGE gel were visualized with Simply Blue Safestain (Invitrogen). For both panels A and B, diabody molecules comprising constructs 10 and 1 , constructs 9 and 1 , and constructs 1 1 and 12A are in lanes 1 , 2 and 3, respectively.
  • FIG.16 BINDING OF DIABODY MOLECULES COMPRISING Fc
  • FIG. 17 SCHEMATIC REPRESENTATION OF POLYPROTEIN
  • Construct ( 13) (SEQ ID NO: 95) comprised, n-VL domain 3G8 - a first linker (GGGSGGGG (SEQ ID NO: 10)) - the VH domain of 2.4G2VH - a second linker (LGGC)- furin recognition site (RAKR (SEQ ID NO: 93))- VL domain of 2.4G2- a third linker (GGGSGGG (SEQ ID NO: 10)- VH domain of 3G8- and a C-terminal LGGC domain; (nucleotide sequence encoding SEQ ID NO: 95 is provided in SEQ ID NO: 96).
  • Construct (14) (SEQ ID NO: 97) comprised, n-VL domain 3G8 - a first linker (GGGSGGGG (SEQ ID NO: 10)) - the VH domain of 2.4G2VH - a second linker (LGGC)- furin recognition site (RAKR (SEQ ID NO: 93))-FMD (Foot and Mouth Disease Virus Protease C3) site-VL domain of 2.4G2- a third linker (GGGSGGG (SEQ ID NO: 10)-VH domain of 3G8- and a C- terminal LGGC domain; (nucleotide sequence encoding SEQ ID NO: 97 is provided in SEQ ID NO: 98).
  • SEQ ID NO: 99 comprised, n-VL domain Hu2B6 - a linker (GGGSGGGG (SEQ ID NO: 10)) - the VH domain of Hu3G8-and a C-terminal FNRGEC (SEQ ID NO: 23) domain; (nucleotide sequence encoding SEQ ID NO: 99 is provided in SEQ ID NO: 100).
  • SEQ ID NO: 101 comprised, n-VL domain Hu3G8 - a linker (GGGSGGGG (SEQ ID NO: 10)) - the VH domain of Hu2B6- and a C-terminal VEPKSC (SEQ ID NO: 77) domain; (nucleotide sequence encoding SEQ ID NO: 101 is provided in SEQ ID NO: 102).
  • FIG. 18 BINDING OF DIABODY MOLECULES DERIVED FROM A
  • FIG.19 BINDING OF DIABODY MOLECULES COMPRISING LAMBDA
  • Diabodies assayed were produced by the recombinant expression system expressing constructs 15 and 16 (SEQ ID NO: 99 and SEQ ID NO:
  • sCD32B was used as the target protein.
  • the secondary probe was
  • FIG. 20 SCHEMATIC REPRESENTATION OF 2B6/4420 DART BOUND
  • the diagram shows the flexibility of the "universal adaptor" anti- fluorescein arm of the DART as well as the possibility of substituting other specificities for the 2B6 arm.
  • V-regions are shown as boxes, GGGSGGGG (SEQ ID NO: 10) linkers are shown as lines, the disulfide bond is shown connecting the two chains.
  • N amino terminus
  • C carboxy terminus
  • FL fluorescein
  • VL light chain variabile region
  • VH heavy chain variable region
  • FIG.21 PANELS A AND B THE 2B6/4420 DART BINDS SPECIFICALLY TO
  • CD32B followed by an antibody specific for CD32B and a secondary detecting antibody conjugated to HRP.
  • B 2B6/4420 or 2B6/3G8 were bound to ELISA plates coated with
  • HulgG or FITC-HuIgG fluorescein-conjugated Binding was detected by engagement with a polyclonal serum specific for 2B6 Fv followed by an HRP-conjugated secondary antibody.
  • FIG. 22 (PANELS A AND B) ACTIVATION OF PURIFIED B CELLS USING
  • B cells were activated using increasing concentrations of anti- human CD79b antibodies FITC-conjugated, CB3.1 -FITC (A) or CB3.2-FITC (B) and 5( ⁇ g/ml of F(ab')2 fragment of GAM IgG Fc specific(x-axis).
  • B cells were activate in the presence of PBS (white bars) or 5 ⁇ / ⁇ of either aFITCaCD32BDART (black bars) or aCD16aCD32BDART (grey bars). The reactions were performed in triplicate and standard deviations were calculated.
  • FIG. 23 (PANELS A AND B) ACTIVATION OF PURIFIED B CELLS
  • the proliferation index was measured in cells activated in the presence of the anti CD79b antibody FITC-conjugated CB3.2-FITC (A) and compared to the proliferation index of cells activated in the presence of the unlabeled CB3.2 antibody (B).
  • FIG. 24 (Upper and Lower Panels) IN VIVO MOUSE B CELL DEPLETION IN
  • mice from MacroGenics breeding colony were injected IV at days 0, 3, 7, 10, 14 and 17 with MGD261 (10, 3, 1 or 0.3mg/kg), or an irrelevant antibody (hE16 lOmg/kg). Blood was collected at days -19 (pre-bleed), 4, 1 1 , 18, 25 and 32 for FACS analysis. Animal health and activity was recorded three times a week.
  • FIG. 25 IN VIVO MOUSE B CELL DEPLETION IN HCD16A/B
  • mice from MacroGenics breeding colony were injected IP at days 0, 2, 4, 7, 9, 1 1, 14, 16 and 18 with 2.4G2-3G8 DB (75ug/mouse), or PBS. Blood was collected at days -10 (pre-bleed), 4, 1 1 and 18 for FACS analysis. Animal health and activity was recorded three times a week.
  • FIG. 26 DEMONSTRATION OF ANTI-TUMOR ACTIVITY OF MGD261
  • mice 250, 25 or 2.5ug MGD261 or with PBS (negative control). Mice were then observed daily and body weight was recorded twice a week. Mice developing hind leg paralysis were sacrificed.
  • FIG. 27 DART EXPRESSION IN A NON-MAMMALIAN HOST
  • FIG. 29 DART-INDUCED HUMAN B-CELL DEATH
  • Apoptosis was assayed by FACS analysis as the percentage of PI + Annexin-V + population of B cells (CD20+ cells) on the total FSC/SSC ungated population.
  • FIG. 30 8B5-CB3.1 DART CONSTRUCTS
  • 8B5-CB3.1 DART constructs were produced to illustrate the present invention.
  • the construct 5 and 6, or 6 and 7, or 8 and 9, or 9and 10, encoded expression plasmids were co-transfected into HEK-293 cells to express 8B5-CB3.1 DART with or without anti flag tag using Lipofectamine 2000 (Invitrogen).
  • the conditioned medium was harvested in every three days for three times.
  • the conditioned medium was then purified using CD32B affinity column.
  • 8B5-CB3.1 DART/ch8B5 competition ELISA were conducted using 96- well Maxisorp plates. After reaction, the plate was washed with PBS-T three times and developed with 80 ⁇ /well of TMB substrate. After 5 minutes incubation, the reaction was stopped by 40 ⁇ /well of 1 % H 2 S0 4 . The OD450 nm was read using a 96-well plate reader and SOFTmax software. The read out was plotted using GraphPadPrism 3.03 software.
  • FIG. 32 SCHEMATIC ILLUSTRATION OF TETRAVALENT DART
  • FIG. 33 Ig-LIKE TETRAVALENT DART
  • FIG. 34 mCD32-hCD16A BINDING ELISA
  • FIG. 35 SCHEMATIC ILLUSTRATION OF Ig DART MOLECULES
  • Ig DART molecules Provides a schematic of Ig DART molecules. Specificity is indicated by shading, pattern or white colored regions, constant regions are shown in black, and disulfide bonds are indicated by dotted black lines.
  • the N-termini of all protein chains are oriented toward the top of the figure, while the C-termini of all protein chains are oriented toward the bottom of the Figure.
  • Illustations A-E are bispecific and Illustations F-J are trispecific.
  • Illustations A and E are tetravalent.
  • Illustations B, C, F, I, and J are hexavalent.
  • Illustations D, G, and H are octavalent. Refer to Figures 1 , 2, 9, 14 and 17 and to Section 3.1 for detailed descriptions of the individual domains.
  • FIG. 36 BINDING ABILITY OF HU2B6 4.5-HU3G8 5.1 BIOSPECIFIC
  • Figure 36 shows the ability of the Hu2B6 4.5-Hu3G8 5.1 biospecific diabody (squares) to bind CD32b and CD 16a relative to Hu2B6 4.5 or Hu3G8 5.1 diabodies (triangles).
  • FIG. 37 SCHEMATIC OF E-COIL AND K-COIL DART DERIVATIVES
  • Figure 37 illustrates the general conformation of E-coil and K-coil DART derivatives.
  • FIG. 38 HELIX ARRANGEMENT OF PREFERRED E-COIL AND K-COIL
  • Figure 38 shows the helix arrangement of preferred '"E-coil" sequence
  • FIG. 39 E-COIL AND K-COIL Fc-CONTAINING DART DERIVATIVES
  • Figure 39 illustrates the different species of E-coil and K-coil Fc- containing DART derivatives that can be formed via chain swapping.
  • FIG. 40 SIZE EXCLUSION CHROMATOGRAPHY ON E-COIL AND/OR K- COIL DERIVATIVES AND E-COIL AND/OR K-COIL FC- CONTAINING DERIVATIVES OF h2B6YAhCB3 DARTS
  • Figure 40 shows the results of size exclusion chromatography on E-coil and/or K-coil derivatives and E-coil and/or K-coil Fc-containing derivatives of
  • h2B6YAhCB3 DARTS Four species of such molecules were analyzed; all had an E-coli and a K-coil: EK (no Fc region), 2.1 mg; EFc/K (Fc linked to E-coil), 2.7 mgs; E/KFc (Fc linked to K-coil), 1.8 mgs; EFc/KFc (Fc linked to the K-coil and the E-coil of the same
  • FIG. 41 STRUCTURE OF PRODUCED DIMER MOLECULES
  • Figure 41 shows the possible structure of the produced dimer molecule identified in the size exclusion chromatograph of Figure 40.
  • FIG. 42 SDS-POLYACRYLAMIDE GEL ELECTROPHORETIC ANALYSIS
  • Figure 42 shows the results of an SDS polyacrylamide gel electrophoretic analysis of the fractions obtained from size exclusion chromatography ( Figure 40) of E- coil and/or K-coil derivatives and E-coil and/or K-coil Fc-containing derivatives of h2B6YAhCB3 DARTs. Flanking lanes: molecular marker controls; Lane 1 : EK (no Fc region); Lane 2: EFc/K, aggregate fraction; Lane 3: EFc/K, monomer fraction; Lane 4:
  • E/KFc aggregate fraction
  • Lane 5 E/KFc, monomer fraction
  • Lane 6 EFc/KFc, aggregate fraction
  • Lane 7 EFc/KFc, dimer fraction
  • Lane 8 EFc/KFc, monomer fraction.
  • FIG. 43 BISPECIFIC BINDING ELISA ANALYSIS OF
  • Figure 43 shows the result of a bispecific binding ELISA comparing E-coil / K-coil Fc-containing h2B6YAhCB3 DART derivatives (EFc/K or E/KFc),
  • FIG. 44 ABILITY OF THE E-COIL AND/OR K-COIL DERIVATIVES AND E-COIL AND/OR K-COIL FC-CONTAINING DERIVATIVES OF h2B6YAhCB3 DARTS TO INHIBIT T-CELL PROLIFERATION
  • Figure 44 shows the ability of the E-coil and/or K-coil derivatives and E- coil and/or K-coil Fc-containing derivatives of h2B6YAhCB3 DARTs to inhibit T-cell proliferation.
  • FIG. 45 hCD16-hCD32B ABD-DART
  • Figure 45 shows a schematic of a recombinant antibody molecule, hCDl 6- hCD32B ABD-DART composed of the the ABD3 domain of streptococcal protein G fused to a recombinant bispecific DART that is immunoreactive with hCD16 and hCD32B antigens.
  • FIG. 46A-46J BINDING AFFINITY OF hCD16-hCD32B ABD-DART
  • ELISA plates were coated with either CD16 antigen (Figure 46A) or human serum albumin (Figure 46B) at a concentration of 2 ⁇ g/mL. Varying concentrations of hCD16-hCD32B ABD-DART ( ⁇ ) and control hCD16-hCD32B DART (o) starting with 2 ⁇ g/mL were bound. Biotinylated sCD32B antigen was added to the plate followed by HRP conjugated Streptavidin for detection. Figure 46C shows that the ABD-DART molecule was capable of simultaneous binding to CD32B, CD16A and HSA.
  • Figures 46D-46I demonstrate that the hCD16-hCD32B ABD-DART and its ABD fusion derivative were found to exhibit equivalent binding soluble CD32B (sCD32B) and to soluble CD16A(158F) (sCD16A).
  • Figure 46 J demonstrates that the DART ABD fusion retained the bi-specific binding of its parental DART, and exhibited binding to sCD16A and CD32B that was unaffected by the presence of human serum albumin.
  • FIG. 47A/47B CYTOTOXICITY OF DART PROTEINS
  • FIG 47A demonstrates PBMC mediated cytotoxicity of DART proteins.
  • ADCC assays were performed using human B-cell lines, Daudi as target cells incubated with PBMC as effector cells. Individual assays were done in triplicate at an effector-to- target ratio of 20: 1 and a titration of antibodies: hCD16A-hCD32B DART ( ⁇ ) and hCD16A-hCD32B ABD DART ( ⁇ ). Cell mediated cytotoxicity was measured by LDH release assay. The lower curve at 10° is hCD 16A-hCD32B ABD DART ( ⁇ ).
  • FIG. 48 IMPROVED PHARMACOKINETIC PROPERTIES OF hCD 16- hCD32B ABD-DART IN C57B1/6 MICE
  • Mouse serum was collected at various time points and concentrations of protein in serum were quantified by ELISA. Pharmacokinetic calculations were performed using WinNonlin
  • FIG. 49A-49C IN VIVO BIOLOGICAL ACTIVITY OF THE ABD-DART
  • FIG. 49A-49C demonstrate that the hCD 16-hCD32B ABD-DART retained and exhibited biological activity, in vivo, after administration to mice.
  • DART molecules having Her2 and T-cell receptor (TCR) binding domains were tested for their ability to mediate cytotoxicity in multiple breast cancer, colon cancer and bladder cancer cell lines that had been previously characterized as exhibiting low levels of HER2 expression (and thus being refractory to treatment with the anti-Her2/neu antibody, Herceptin®.
  • the tested breast cancer cell lines are ZR75-1 (HER2 2+) (FIG.
  • FIG. 50A MCF-7 (HER2 1 +)
  • FIG. 50B MCF-7 (HER2 1 +)
  • FIG. 50C MDA-MB468 (HER2-ve)
  • the non-breast cancer cell lines tested are HT-29 (colon cancer cell line) (FIG. 50D) and
  • SW780 (bladder cancer cell line) (FIG. 50E).
  • FIG. 51A-51B PURIFICATION OF KYK2VL-4420VH-GFNRGEC (SEQ ID NO: 313)
  • FIG. 51A-51 B show the purification of KYK2VL-4420VH-GFNRGEC (SEQ ID NO: 313) - E Coil And 4420VL-KYK2VH-GVEPKSC (Seq Id No: 314) polypeptides.
  • FIG. 52 BINDING SPECIFICITY OF KYK2-4420 VF DART
  • FIG. 52 shows the binding specificity of KYK2-4420 VF DART.
  • FIG. 53 CONSTRUCTION OF PLASMID pPAL7 COIL-ABD
  • FIG. 53 shows a schematic description of the construction of plasmid pPAL7 Kcoil -ABD.
  • FIG. 54 WESTERN BLOT ANALYSIS OF KCOIL ABD PROTEIN PURIFIED
  • Figure 54 shows a Western blot analysis of Kcoil ABD protein purified by the PROFINITY EXACTTM purification resin (small scale from 10 ml culture). Lanes 1 and 5: crude lysates; lanes 2 and 6: flow through, lanes 3, 4, 8, and 9: purified protein; lane 7: Marker. Primary antibody: Rabbit polyclonal anti-EK antibody; Secondary Antibody: anti-rabbit HRP.
  • FIG. 55 SDS-PAGE ANALYSIS OF KCOIL ABD PROTEIN PURIFIED BY THE
  • Figure 55 shows the results of SDS-PAGE analysis on the fractions eluted from the PROFINITY EXACTTM purification resin.
  • L Crude E. coli lysate
  • FT Flow through
  • W Washing
  • M Marker.
  • FIG. 56A-56C KYK2 4420 EK ABD DART BINDING BY DUAL AFFINITY ELISA
  • Figures 56A-56C show ELISA results that demonstrate the KYK2-4420
  • FIG. 56A DARTs captured with NKG2D Fc and detected with biotinylated monoclonal anti-EK antibody.
  • Figure 56B DARTs captured with monoclonal anti-EK antibody and detected with HRP-human albumin.
  • Figure 56C DARTs captured with NKG2D Fc and detected with biotinylated anti-FITC antibody. The ELISA results show that all of the domains in the complex can bind to their respective ligands.
  • Each polypeptide chain of the diabody molecule comprises a VL domain and a VH domain, which are covalently linked such that the domains are constrained from self assembly. Interaction of two of the polypeptide chains will produce two VL-VH pairings, forming two eptipoe binding sites, i.e. , a bivalent molecule. Neither the VH or VL domain is constrained to any position within the polypeptide chain, i.e., restricted to the amino (N) or carboxy (C) teminus, nor are the domains restricted in their relative positions to one another, i. e., the VL domain may be N-terminal to the VH domain and vice-versa.
  • each polypeptide will comprise a VH A and a VL A .
  • Homodimerization of two polypeptide chains of the antibody will result in the formation two VL A -VH A binding sites, resulting in a bivalent monospecific antibody.
  • VL and VH domains are derived from antibodies specific for different antigens
  • formation of a functional bispecific diabody requires the interaction of two different polypeptide chains, i.e. , formation of a heterodimer.
  • one polypeptide chain will comprise a VL A and a VL B ;
  • one or more of the polypeptide chains of the diabody comprises an Fc domain.
  • Fc domains in the polypeptide chains of the diabody molecules preferentially dimerize, resulting in the formation of a diabody molecule that exhibits immunoglobulin-like properties, e.g. , Fc-FcyR, interactions.
  • Fc comprising diabodies may be dimers, e.g. , comprised of two polypeptide chains, each comprising a VH domain, a VL domain and an Fc domain. Dimerization of said polypeptide chains results in a bivalent diabody comprising an Fc domain, albeit with a structure distinct from that of an unmodified bivalent antibody (FIG.1 1 ).
  • diabody molecules will exhibit altered phenotypes relative to a wild-type immunoglobulin, e.g. , altered serum half-life, binding properties, etc.
  • diabody molecules comprising Fc domains may be tetramers.
  • Such tertramers comprise two 'heavier' polypeptide chains, i.e. a polypeptide chain comprising a VL, aVH and an Fc domain, and two 'lighter' polypeptide chains, i.e., polypeptide chain comprising a VL and a VH. Said lighter and heavier chains interact to form a monomer, and said monomers interact via their unpaired Fc domains to form an Ig-like molecule.
  • Such an Ig-like diabody is tetravalent and may be
  • the at least two binding sites of the diabody molecule can recognize the same or different epitopes.
  • Different epitopes can be from the same antigen or epitopes from different antigens.
  • the epitopes are from different cells.
  • the epitopes are cell surface antigens on the same cell or virus.
  • the epitopes binding sites can recognize any antigen to which an antibody can be generated. For example, proteins, nucleic acids, bacterial toxins, cell surface markers, autoimmune markers, viral proteins, drugs, etc.
  • At least one epitope binding site of the diabody is specific for an antigen on a particular cell, such as a B-cell or T-cell, a phagocytotic cell, a natural killer (NK) cell or a dendritic cell.
  • a particular cell such as a B-cell or T-cell, a phagocytotic cell, a natural killer (NK) cell or a dendritic cell.
  • Each domain of the polypeptide chain of the diabody i.e. , the VL, VH and FC domain may be separated by a peptide linker.
  • the peptide linker may be 0, 1 , 2, 3, 4, 5, 6, 7, 8, or 9. amino acids.
  • the amino acid linker sequence is GGGSGGGG (SEQ ID NO: 10) encoded by the nucleic acid sequence (SEQ ID NO: 74).
  • each polypeptide chain of the diabody molecule is engineered to comprise at least one cysteine residue that will interact with a counterpart at least one cysteine residue on a second polypeptide chain of the invention to form an interchain disulfide bond.
  • Said interchain disulfide bonds serve to stabilize the diabody molecule, improving expression and recovery in recombinant systems, resulting in a stable and consistent formulation as well as improving the stability of the isolated and/or purified product in vivo.
  • Said at least one cysteine residue may be introduced as a single amino acid or as part of larger amino-acid sequence, e.g. hinge domain, in any portion of the polypeptide chain.
  • said at least one cysteine residue is engineered to occur at the C-terminus of the polypeptide chain.
  • said at least one cysteine residue in introduced into the polypeptide chain within the amino acid sequence LGGC.
  • the C-terminus of the polypeptide chain comprising the diabody molecule of the invention comprises the amino acid sequence LGGC.
  • said at least one cysteine residue is introduced into the polypeptide within an amino acid sequence comprising a hinge domain, e.g. SEQ ID NO: 1 or SEQ ID NO: 4.
  • the C-terminus of a polypeptide chain of the diabody molecule of the invention comprises the amino acid sequence of an IgG hinge domain, e.g.
  • the C-terminus of a polypeptide chain of a diabody molecule of the invention comprises the amino acid sequence VEPKSC (SEQ ID NO: 77), which can be encoded by nucleotide sequence (SEQ ID NO: 78).
  • the C- terminus of a polypeptide chain comprising the diabody of the invention comprises the amino acid sequence LGGCFNRGEC (SEQ ID NO: 17), which can be encoded by the nucleotide sequence (SEQ ID NO: 76).
  • the C-terminus of a polypeptide chain comprising the diabody of the invention comprises the amino acid sequence FNRGEC (SEQ ID NO: 23), which can be encoded by the nucleotide sequence (SEQ ID NO: 75).
  • the diabody molecule comprises at least two polypeptide chains, each of which comprise the amino acid sequence LGGC and are covalently linked by a disulfide bond between the cysteine residues in said LGGC sequences.
  • the diabody molecule comprises at least two polypeptide chains, one of which comprises the sequence FNRGEC (SEQ ID NO: 23) while the other comprises a hinge domain (containing at least one cysteine residue), wherein said at least two polypeptide chains are covalently linked by a disulfide bond between the cysteine residue in FNRGEC (SEQ ID NO: 23) and a cysteine residue in the hinge domain.
  • cysteine residue responsible for the disulfide bond located in the hinge domain is Cys-128 (as numbered according to Kabat EU; located in the hinge domain of an unmodified, intact IgG heavy chain) and the counterpart cysteine residue in SEQ ID NO: 23 is Cys-214 (as numbered according to Kabat EU; located at the C-terminus of an unmodified, intact IgG light chain) (Elkabetz et al. (2005) " Cysteines In CHI Underlie Retention Of Unassembled Ig Heavy Chains, " J. Biol. Chem. 280: 14402- 14412; hereby incorporated by reference herein in its entirety).
  • the at least one cysteine residue is engineered to occur at the N-terminus of the amino acid chain. In still other embodiments, the at least one cysteine residue is engineered to occur in the linker portion of the polypeptide chain of the diabody molecule. In further embodiments, the VH or VL domain is engineered to comprise at least one amino acid modification relative to the parental VH or VL domain such that said amino acid modification comprises a substitution of a parental amino acid with cysteine.
  • the invention encompasses diabody molecules comprising an Fc domain or portion thereof (e.g. a CH2 domain, or CH3 domain).
  • the Fc domain or portion thereof may be derived from any immunoglobulin isotype or allotype including, but not limited to, IgA, IgD, IgG, IgE and IgM.
  • the Fc domain (or portion thereof) is derived from IgG.
  • the IgG isotype is IgGl , IgG2, IgG3 or IgG4 or an allotype thereof.
  • the diabody molecule comprises an Fc domain, which Fc domain comprises a CH2 domain and CH3 domain independently selected from any immunoglobulin isotype (i.e. an Fc domain comprising the CH2 domain derived from IgG and the CH3 domain derived form IgE, or the CH2 domain derived from IgGl and the CH3 domain derived from IgG2, etc.).
  • Said Fc domain may be engineered into a polypeptide chain comprising the diabody molecule of the invention in any position relative to other domains or portions of said polypeptide chain (e.g.
  • the Fc domain, or portion thereof may be c-terminal to both the VL and VH domains of the polypeptide of the chain; may be n-terminal to both the VL and VH domains; or may be N-terminal to one domain and c-terminal to another (i.e. , between two domains of the polypeptide chain)).
  • the present invention also encompasses molecules comprising a hinge domain.
  • the hinge domain be derived from any immunoglobulin isotype or allotype including IgA, IgD, IgG, IgE and IgM.
  • the hinge domain is derived from IgG, wherein the IgG isotype is IgG l , IgG2, IgG3 or IgG4, or an allotpye thereof.
  • Said hinge domain may be engineered into a polypeptide chain comprising the diabody molecule together with an Fc domain such that the diabody molecule comprises a hinge-Fc domain.
  • the hinge and Fc domain are independently selected from any immunoglobulin isotype known in the art or exemplified herein. In other embodiments the hinge and Fc domain are separated by at least one other domain of the polypeptide chain, e.g. , the VL domain.
  • the hinge domain, or optionally the hinge-Fc domain may be engineered in to a polypeptide of the invention in any position relative to other domains or portions of said polypeptide chain.
  • a polypeptide chain of the invention comprises a hinge domain, which hinge domain is at the C-terminus of the polypeptide chain, wherein said polypeptide chain does not comprise an Fc domain.
  • a polypeptide chain of the invention comprises a hinge-Fc domain, which hinge-Fc domain is at the C-terminus of the polypeptide chain. In further embodiments, a polypeptide chain of the invention comprises a hinge-Fc domain, which hinge-Fc domain is at the N-terminus of the polypeptide chain.
  • the invention encompasses multimers of polypeptide chains, each of which polypeptide chains comprise a VH and VL domain.
  • the polypeptide chains in said multimers further comprise an Fc domain.
  • the VL and VH domains comprising each polypeptide chain have the same specificity, and said diabody molecule is bivalent and monospecific. In other embodiments, the VL and VH domains comprising each polypeptide chain have differing specificity and the diabody is bivalent and bispecific.
  • diabody molecules of the invention encompass tetramers of polypeptide chains, each of which polypeptide chain comprises a VH and VL domain.
  • two polypeptide chains of the tetramer further comprise an Fc domain.
  • the tetramer is therefore comprised of two 'heavier' polypeptide chains, each comprising a VL, VH and Fc domain, and two 'lighter' polypeptide chains, comprising a VL and VH domain.
  • tetravalent immunoglobulin-like molecule Interaction of a heavier and lighter chain into a bivalent monomer coupled with dimerization of said monomers via the Fc domains of the heavier chains will lead to formation of a tetravalent immunoglobulin-like molecule (exemplified in Example 6.2 and Example 6.3).
  • the monomers are the same, and the tetravalent diabody molecule is monospecific or bispecific. In other aspects the monomers are different, and the tetra valent molecule is bispecific or tetraspecific.
  • Formation of a tetraspecific diabody molecule as described supra requires the interaction of four differing polypeptide chains. Such interactions are difficult to achieve with efficiency within a single cell recombinant production system, due to the many variants of potential chain mispairings.
  • One solution to increase the probability of mispairings is to engineer "knobs-into-holes" type mutations into the desired polypeptide chain pairs. Such mutations favor heterodimerization over homodimerization.
  • an amino acid substitution (preferably a substitution with an amino acid comprising a bulky side group forming a 'knob', e.g., tryptophan) can be introduced into the CH2 or CH3 domain such that steric interference will prevent interaction with a similarly mutated domain and will obligate the mutated domain to pair with a domain into which a complementary, or accommodating mutation has been engineered, i.e., 'the hole' (e.g., a substitution with glycine).
  • Such sets of mutations can be engineered into any pair of polypeptides comprising the diabody molecule, and further, engineered into any portion of the polypeptides chains of said pair.
  • the invention also encompasses diabody molecules comprising variant Fc or variant hinge-Fc domains (or portion thereof), which variant Fc domain comprises at least one amino acid modification (e.g. substitution, insertion deletion) relative to a comparable wild-type Fc domain or hinge-Fc domain (or portion thereof).
  • Molecules comprising variant Fc domains or hinge-Fc domains (or portion thereof) e.g. , antibodies
  • the variant phenotype may be expressed as altered serum half-life, altered stability, altered susceptibility to cellular enzymes or altered effector function as assayed in an NK dependent or macrophage dependent assay.
  • Fc domain variants identified as altering effector function are disclosed in International Application WO04/063351 , U.S. Patent Application Publications 2005/0037000 and 2005/0064514, U.S. Provisional Applications 60/626,510. filed November 10, 2004, 60/636,663, filed December 15, 2004, and 60/781 ,564, filed March 10, 2006, and U.S. Patent Applications 1 1/271 , 140, filed November 10, 2005, and 1 1/305,787, filed
  • the bispecific diabodies of the invention can simultaneously bind two separate and distinct epitopes.
  • the epitopes are from the same antigen.
  • the epitopes are from different antigens.
  • at least one epitope binding site is specific for a determinant expressed on an immune effector cell (e.g. CD3, CD 16, CD32, CD64, etc.) which are expressed on T lymphocytes, natural killer (NK) cells or other mononuclear cells.
  • the diabody molecule binds to the effector cell determinant and also activates said effector cell.
  • diabody molecules of the invention may exhibit Ig-like functionality independent of whether they further comprise an Fc domain (e.g., as assayed in any effector function assay known in the art or exemplified herein (e.g., ADCC assay).
  • the bispecific diabody of the invention binds both a cancer antigen on a tumor cell and an effector cell determinant while activating said cell.
  • the bispecific diabody or diabody molecule of the invention may inhibit activation of a target, e.g., effector, cell by simultaneously binding, and thus linking, an activating and inhibitory receptor on the same cell (e.g., bind both CD32A and CD32B, BCR and CD32B, or IgERI and CD32B) as described supra (see, Background Section).
  • the bispecific diabody may exhibit anti-viral properties by simultaneously binding two neutralizing epitopes on a virus (e.g., RSV epitopes; WNV epitopes such as El 6 and E53).
  • bispecific diabody molecules of the invention offer unique opportunities to target specific cell types.
  • the bispecific diabody or diabody molecule can be engineered to comprise a combination of epitope binding sites that recognize a set of antigens unique to a target cell or tissue type.
  • low affinity biding domains can be used to construct the diabody or diabody molecule. Such low affinity binding domains will be unable to bind to the individual epitope or antigen with sufficient avidity for therapeutic purposes.
  • the avidity of the diabody or diabody molecule for the cell or tissue, relative to a cell or tissue expressing only one of the antigens, will be increased such that said cell or tissue can be effectively targeted by the invention.
  • Such a bispecific molecule can exhibit enhanced binding to one or both of its target antigens on cells expressing both of said antigens relative to a monospecific diabody or an antibody with a specificity to only one of the antigens.
  • the binding properties of the diabodies of the invention are characterized by in vitro functional assays for determining binding activity and/or one or more FcyR mediator effector cell functions (mediated via Fc-FcyR interactions or by the immunospecific binding of a diabody molecule to an FcyR) (See Section 5.4.2 and 5.4.3).
  • the affinities and binding properties of the molecules e.g.
  • diabodies, of the invention for an FcyR can be determined using in vitro assays (biochemical or immunological based assays) known in the art for determining binding domain-antigen or Fc-FcyR interactions, i.e., specific binding of an antigen to a binding domain or specific binding of an Fc region to an FcyR, respectively, including but not limited to ELISA assay, surface plasmon resonance assay, immunoprecipitation assays (See Section 5.4.2).
  • the molecules of the invention have similar binding properties in in vivo models (such as those described and disclosed herein) as those in in vitro based assays.
  • the present invention does not exclude molecules of the invention that do not exhibit the desired phenotype in in vitro based assays but do exhibit the desired phenotype in vivo.
  • molecules of the invention are engineered to comprise an altered glycosylation pattern or an altered glycoform relative to the comparable portion of the template molecule.
  • Engineered glycoforms may be useful for a variety of purposes, including, but not limited to, enhancing effector function.
  • Engineered glycoforms may be generated by any method known to one skilled in the art, for example by using engineered or variant expression strains, by co-expression with one or more enzymes, for example, DI N-acetylglucosaminyltransferase III (GnTIl 1), by expressing a diabody of the invention in various organisms or cell lines from various organisms, or by modifying carbohydrate(s) after the diabody has been expressed and purified.
  • GnTIl 1 DI N-acetylglucosaminyltransferase III
  • Oligosaccharide Improves Binding To Human Fc gamma RIII And Antibody-Dependent Cellular Toxicity, " J Biol Chem 277:26733-26740; Shinkawa e/ al. (2003) "The Absence Of Fucose But Not The Presence Of Galactose Or Bisecting N-Acetylglucosamine Of Human IgGl Complex-Type Oligosaccharides Shows The Critical Role Of Enhancing Antibody-Dependent Cellular Cytotoxicity, " J Biol Chem 278:3466-3473) US 6,602,684; USSN 10/277,370; USSN 10/1 13,929; PCT WO 00/61739A1 ; PCT WO 01/292246A1 ; PCT WO 02/31 1 140A1 ; PCT WO 02/30954A1 ; PotillegentTM technology (Biowa, Inc.
  • GlycoMAbTM glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland); each of which is incorporated herein by reference in its entirety. See, e.g., WO 00061739; EA01229125; US 200301 15614;
  • the invention further encompasses incorporation of unnatural amino acids to generate the diabodies of the invention.
  • Such methods are known to those skilled in the art such as those using the natural biosynthetic machinery to allow incorporation of unnatural amino acids into proteins, see, e.g. , Wang et al. (2002) “Expanding The Genetic Code, " Chem. Comm. 1 : 1 -1 1 ; Wang et al. (2001) “Expanding The Genetic Code Of Escherichia coli, " Science, 292: 498-500; van Hest et al. (2001 ) "Protein-Based
  • the invention encompasses methods of modifying a VL, VH or Fc domain of a molecule of the invention by adding or deleting a glycosylation site.
  • Methods for modifying the carbohydrate of proteins are well known in the art and encompassed within the invention, see, e.g. , U.S. Patent No. 6,218,149; EP 0 359 096 Bl ; U.S. Publication No. US 2002/0028486; WO 03/035835; U.S. Publication No.
  • the diabody molecules of the present invention may be constructed to comprise a domain that is a binding ligand for the Natural Killer Group 2D (NKG2D) receptor.
  • NSG2D Natural Killer Group 2D
  • binding ligands and particularly those which are not expressed on normal cells, include the histocompatibility 60 (H60) molecule, the product of the retinoic acid early inducible gene-1 (RAE-1 ), and the murine UL16-binding proteinlike transcript 1 (MULT1 ) (Raulet D.H.
  • the sequence of MICA is SEQ ID NO: 311 :
  • the sequence of MICB is SEQ ID NO: 312:
  • Antibodies that specifically bind to the ⁇ -cell Receptor include the anti- TCR antibody BMA 031 (Kurrle, R. et al. ( 1989) "BMA 031 - A TCR-Specific
  • Antibodies Directed Against The Human ⁇ / ⁇ T Cell Receptor “ J. Immunol. 147(12):4366- 4373).
  • Antibodies that specifically bind to the NKG2D Receptor include KYK-2.0 (Kwong, KY et al. (2008) "Generation, Affinity Maturation, And Characterization Of A Human Anti-Human NKG2D Monoclonal Antibody With Dual Antagonistic And Agonistic Activity ' J. Mol. Biol. 384: 1 143-1 156; and PCT/US09/5491 1 ).
  • the target cell is now redirected to be a cell that can be bound by cells that array the (NKG2D) receptor.
  • the NKG2D receptor is expressed on all human (and other mammalian) Natural Killer cells (Bauer, S. et al. (1999) "Activation OfNK Cells And T Cells By NKG2D, A Receptor For Stress-Inducible MICA," Science 285(5428):727-729; Jamieson, A.M. et al.
  • the diabody molecules of the present invention may be constructed to comprise a domain that is a binding ligand for the T-cell receptor ("TCR").
  • TCR T-cell receptor
  • the TCR is natively expressed by CD4+ or CD8+ T-cells, and permits such cells to recognize antigenic peptides that are bound and presented by class I or class II MHC proteins of antigen-presenting cells.
  • Recognition of a pMHC (peptide-MHC) complex by a TCR initiates the propagation of a cellular immune response that leads to the production of cytokines and the lysis of the antigen-presenting cell (see, e.g. , Armstrong, K.M. et al.
  • such diabody molecules By constructing such diabody molecules to additionally comprise at least one epitope-binding domain capable of binding to, for example, a receptor present on the surface of a target cell, such diabody molecules will be DART molecules and thus be capable of binding to the target cells and thereby cause the target cells to display the binding ligand for the Natural Killer Group 2D (NKG2D) receptor or to the TCR
  • NSG2D Natural Killer Group 2D
  • Such diabodies can be used to redirect any desired target cell into a cell that is a target of NK cell-mediated cell lysis or T-cell mediated cytotoxicity.
  • the epitope-binding domain of the diabody capable of binding to a receptor present on the surface of a target cell is an epitope that binds to a tumor-associated antigen so as to redirect such cancer cells into substrates for NK cell-mediated cell lysis or T-cell mediated cytotoxicity.
  • tumor-associated antigens that is a breast cancer antigen, an ovarian cancer antigen, a prostate cancer antigen, a cervical cancer antigen, a pancreatic carcinoma antigen, a lung cancer antigen, a bladder cancer antigen, a colon cancer antigen, a testicular cancer antigen, a glioblastoma cancer antigen, an antigen associated with a B cell malignancy, an antigen associated with multiple myeloma, an antigen associated with non-Hodgkins lymphoma, or an antigen associated with chronic lymphocytic leukemia.
  • Suitable tumor-associated antigens for such use include A33 (a colorectal carcinoma antigen; Almqvist, Y. 2006, Nucl Med Biol. Nov;33(8):991 -998); B l (Egloff, A.M. et al. 2006, Cancer Res. 66(l ):6-9); BAGE (Bodey, B. 2002 Expert Opin Biol Ther. 2(6):577-84); beta-catenin (Prange W. et al. 2003 J Pathol. 201 (2):250-9); CA125 (Bast, R.C. Jr. et al. 2005 Int J Gynecol Cancer 15 Suppl 3:274-81 ); CD5 (Calin, G.A.
  • CD22 Kerman, R.J. 2006 AAPS J. 18;8(3):E532-51
  • CD23 Rosati, S. et al. 2005 Curr Top Microbiol Immunol. 5;294:91 -107
  • CD25 Teroussard, X. et al. 1998 Hematol Cell Ther. 40(4): 139-48
  • CD27 Bataille, R. 2006 Haematologica 91 (9): 1234- 40
  • CD28 Breast, R. 2006 Haematologica 91 (9): 1234-40
  • CD36 Ga, Y. 2005 Lab Hematol.
  • CD40/CD154 Messmer, D. et al. 2005 Ann N YAcad Sci. 1062:51 - 60
  • CD45 Jurcic, J.G. 2005 Curr Oncol Rep. 7(5):339-46
  • CD56 Bataille, R. 2006 Haematologica 91(9): 1234-40
  • CD79a/CD79b Troussard, X. et al. 1998 Hematol Cell Ther. 40(4): 139-48; Chu, P.G. et al. 2001 Appl Immunohistochem Mol Morphol. 9(2):97- 106
  • CD 103 Troussard, X. et al.
  • CDK4 Lee, Y.M. et al. 2006 Cell Cycle 5(18):21 10-4
  • CEA carcinoembryonic antigen
  • Tellez-Avila F.I. et al. 2005 Rev Invest Clin. 57(6):814-9
  • CTLA4 Peggs, K.S. et al. 2006 Curr Opin Immunol. 18(2):206-13
  • EGF-R epidermal growth factor receptor; Adenis, A. et al. 2003 Bull Cancer.
  • MUM-1 (Castelli, C. et al. 2000 J Cell Physiol. 182(3):323-31); N-acetylglucosaminyltransferase (Dennis, J.W. 1999 Biochim Biophys Acta. 6; 1473(1 ):21 -34); pl 5 (Gil, J. et al. 2006 Nat Rev Mol Cell Biol. 7(9):667-77); PSA (prostate specific antigen; Cracco, CM. et al. 2005 Minerva Urol Nefrol. 57(4):301-1 1); PSMA (Ragupathi, G. 2005 Cancer Treat Res.
  • TNF-receptor TNF-a receptor, TNF- ⁇ receptor; or TNF- ⁇ receptor
  • van Horssen R. et al. 2006 Oncologist. 1 1 (4):397-408; Gardnerova, M. et al. 2000 Curr Drug Targets. l (4):327-64
  • VEGF receptor O'Dwyer. P.J. 2006 Oncologist. 1 1(9):992-8).
  • ADAM-9 United States Patent Publication No. 2006/0172350; PCT Publication No. WO 06/084075;
  • Oncostatin M Oncostatin Receptor Beta
  • PIPA United States Patent No. 7,405,061 ; PCT
  • antigens specific to particular infectious agents e.g., viral agents including, but not limited to human immunodeficiency virus (HIV), hepatitis B virus (HBV), influenza, human papilloma virus (HPV), foot and mouth
  • infectious agents e.g., viral agents including, but not limited to human immunodeficiency virus (HIV), hepatitis B virus (HBV), influenza, human papilloma virus (HPV), foot and mouth
  • coxsackieviruses the rabies virus, herpes simplex virus (HSV), and the causative agents of gastroenteritis, including rotaviruses, adenoviruses, caliciviruses, astroviruses and Norwalk virus; bacterial agents including, but not limited to E. coli, Salmonella thyphimurium, Pseudomonas aeruginosa, Vibrio cholerae.
  • Neisseria gonorrhoeae Helicobacter pylori, Hemophilus influenzae, Shigella dysenteriae, Staphylococcus aureus, Mycobacterium tuberculosis and Streptococcus pneumoniae, fungal agents and parasites such as Giardi.
  • such epitope may bind to an Fc receptor (e.g. , FcyRI or FcyRII), so as to, for example redirect acute monocytic leukemic cells into substrates for NK cell-mediated cell lysis.
  • Fc receptor e.g. , FcyRI or FcyRII
  • the diabodies of the present invention comprise antigen binding domains generally derived from immunoglobulins or antibodies.
  • the antibodies from which the binding domains used in the methods of the invention are derived may be from any animal origin including birds and mammals (e.g. , human, non-human primate, murine, donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken).
  • the antibodies are human or humanized monoclonal antibodies.
  • "human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or libraries of synthetic human immunoglobulin coding sequences or from mice that express antibodies from human genes.
  • the invention contemplates the use of any antibodies known in the art for the treatment and/or prevention of cancer, autoimmune disease, inflammatory disease or infectious disease as source of binding domains for the diabodies of the invention.
  • Non- limiting examples of known cancer antibodies are provided in section 5.7.1 as well as other antibodies specific for the listed target antigens and antibodies against the cancer antigens listed in section 5.6.1 ; nonlimiting examples of known antibodies for the treatment and/or prevention of autoimmune disease and inflammatory disease are provided in section 5.7.2. as well as antibodies against the listed target antigens and antibodies against the antigens listed in section 5.6.2; in other embodiments antibodies against epitopes associated with infectious diseases as listed in Section 5.6.3 can be used.
  • the antibodies comprise a variant Fc region comprising one or more amino acid modifications, which have been identified by the methods of the invention to have a conferred effector function and/or enhanced affinity for FcyRIIB and a decreased affinity for FcyRIIIA relative to a comparable molecule comprising a wild type Fc region.
  • a non-limiting example of the antibodies that are used for the treatment or prevention of inflammatory disorders which can be engineered according to the invention is presented in Table 9, and a non-limiting example of the antibodies that are used for the treatment or prevention of autoimmune disorder is presented in Table 10.
  • variable domains derived from human, chimeric or humanized antibodies are particularly desirable for therapeutic treatment of human subjects.
  • Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also U.S. Patent Nos. 4,444,887 and 4,716,1 1 1 ; and International
  • a humanized antibody is an antibody, a variant or a fragment thereof which is capable of binding to a predetermined antigen and which comprises a framework region having substantially the amino acid sequence of a human immunoglobulin and a CDR having substantially the amino acid sequence of a non-human immunoglobulin.
  • a humanized antibody may comprise substantially all of at least one, and typically two, variable domains in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.
  • the framework and CDR regions of a humanized antibody need not correspond precisely to the parental sequences, e.g., the donor CDR or the consensus framework may be mutagenized by substitution, insertion or deletion of at least one residue so that the CDR or framework residue at that site does not correspond to either the consensus or the donor antibody. Such mutations, however, are preferably not extensive. Usually, at least 75% of the humanized antibody residues will correspond to those of the parental framework region (FR) and CDR sequences, more often 90%, and most preferably greater than 95%. Humanized antibodies can be produced using variety of techniques known in the art, including but not limited to, CDR-grafting (European Patent No. EP 239,400; International Publication No. WO 91/09967; and U.S. Patent Nos.
  • framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding.
  • framework substitutions are identified by methods well known in the art, e.g. , by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g. , Queen et al., U.S. Patent No.
  • the humanized binding domain specifically binds to the same epitope as the donor murine antibody.
  • the donor and acceptor antibodies may be derived from animals of the same species and even same antibody class or sub-class. More usually, however, the donor and acceptor antibodies are derived from animals of different species.
  • the donor antibody is a non-human antibody, such as a rodent mAb, and the acceptor antibody is a human antibody.
  • At least one CDR from the donor antibody is grafted onto the human antibody.
  • at least two and preferably all three CDRs of each of the heavy and/or light chain variable regions are grafted onto the human antibody.
  • the CDRs may comprise the Kabat CDRs, the structural loop CDRs or a combination thereof.
  • the invention encompasses a humanized FcyRIIB antibody comprising at least one CDR grafted heavy chain and at least one CDR- grafted light chain.
  • the diabodies used in the methods of the invention include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the diabody.
  • the diabody derivatives include diabodies that have been modified, e.g. , by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
  • a chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules such as antibodies having a variable region derived from a non-human antibody and a human immunoglobulin constant region.
  • Methods for producing chimeric antibodies are known in the art. See e.g., Morrison (1985) "Transfectomas Provide Novel Chimeric Antibodies, " Science 229: 1202-1207; Oi et al. (1986) "Chimeric Antibodies, " BioTechniques 4:214-221 ; Gillies et al. (1989) "High-Level Expression Of Chimeric Antibodies Using Adapted cDNA Variable Region Cassettes, " J. Immunol. Methods 125: 191 -202; and U.S. Patent Nos. 6,31 1,415, 5,807,715, 4,816,567, and 4,816,397, which are incorporated herein by reference in their entirety.
  • framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding.
  • These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., U.S. Patent No. 5,585,089; and Riechmann et al.
  • Monoclonal antibodies from which binding domains of the diabodies of the invention can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al. , Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.
  • the term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
  • the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
  • mice can be immunized with an antigen of interest or a cell expressing such an antigen. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells.
  • Hybridomas are selected and cloned by limiting dilution.
  • the hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding the antigen.
  • Ascites fluid which generally contains high levels of antibodies, can be generated by inoculating mice intraperitoneally with positive hybridoma clones.
  • Antigens of interest include, but are not limited to, antigens associated with the cancers provided in section 5.8.1 , antigens associated with the autoimmune diseases and inflammatory diseases provided in section 5.8.2, antigens associated with the infectious diseases provided in section 5.8.3, and the toxins provided in section 5.8.4.
  • Antibodies can also be generated using various phage display methods known in the art.
  • phage display methods functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
  • phage can be utilized to display antigen binding domains, such as Fab and Fv or disulfide-bond stabilized Fv, expressed from a repertoire or combinatorial antibody library ⁇ e.g., human or murine).
  • Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g. , using labeled antigen or antigen bound or captured to a solid surface or bead.
  • Phage used in these methods are typically filamentous phage, including fd and Ml 3.
  • the antigen binding domains are expressed as a recombinantly fused protein to either the phage gene III or gene VIII protein.
  • Examples of phage display methods that can be used to make the immunoglobulins, or fragments thereof, of the present invention include those disclosed in Brinkmann et al. (1995) "Phage Display Of Disulfide-Stabilized Fv Fragments, " J.
  • Phage display technology can be used to increase the affinity of an antibody for its antigen. This technique would be useful in obtaining high affinity antibodies.
  • the technology referred to as affinity maturation, employs mutagenesis or CDR walking and re-selection using the cognate antigen to identify antibodies that bind with higher affinity to the antigen when compared with the initial or parental antibody (See, e.g.,Glaser et al. (1992) "Dissection Of The Combining Site In A Humanized Ant i- Tac Antibody, " J. Immunology 149:2607-2614). Mutagenizing entire codons rather than single nucleotides results in a semi-randomized repertoire of amino acid mutations.
  • Libraries can be constructed consisting of a pool of variant clones each of which differs by a single amino acid alteration in a single CDR and which contain variants representing each possible amino acid substitution for each CDR residue.
  • Mutants with increased binding affinity for the antigen can be screened by contacting the immobilized mutants with labeled antigen. Any screening method known in the art can be used to identify mutant antibodies with increased avidity to the antigen (e.g., ELISA) (See Wu et al.
  • the present invention also encompasses the use of binding domains comprising the amino acid sequence of any of the binding domains described herein or known in the art with mutations ⁇ e.g., one or more amino acid substitutions) in the framework or CDR regions.
  • mutations in these binding domains maintain or enhance the avidity and/or affinity of the binding domains for FcyRIIB to which they immunospecifically bind.
  • Standard techniques known to those skilled in the art e.g., immunoassays can be used to assay the affinity of an antibody for a particular antigen.
  • Standard techniques known to those skilled in the art can be used to introduce mutations in the nucleotide sequence encoding an antibody, or fragment thereof, including, e.g., site-directed mutagenesis and PCR-mediated mutagenesis, which results in amino acid substitutions.
  • the derivatives include less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the original antibody or fragment thereof.
  • the derivatives have conservative amino acid substitutions made at one or more predicted non-essential amino acid residues.
  • At least one of the binding domains of the diabodies of the invention agonizes at least one activity of FcyRIIB.
  • said activity is inhibition of B cell receptor-mediated signaling.
  • the binding domain inhibits activation of B cells, B cell proliferation, antibody production, intracellular calcium influx of B cells, cell cycle progression, or activity of one or more downstream signaling molecules in the FcyRIIB signal
  • the binding domain enhances phosphorylation of FcyRIIB or SHIP recruitment.
  • the binding domain inhibits MAP kinase activity or Akt recruitment in the B cell receptor-mediated signaling pathway.
  • the binding domain agonizes FcyRIIB-mediated inhibition of FCERI signaling.
  • said binding domain inhibits FcsRI-induced mast cell activation, calcium mobilization, degranulation, cytokine production, or serotonin release.
  • the binding domains of the invention stimulate phosphorylation of FcyRIIB, stimulate recruitment of SHIP, stimulate SHIP phosphorylation and its association with She, or inhibit activation of MAP kinase family members (e.g., Erkl , Erk2, J K, p38, etc.).
  • the binding domains of the invention enhance tyrosine phosphorylation of p62dok and its association with SHIP and rasGAP.
  • the binding domains of the invention inhibit FcyR-mediated phagocytosis in monocytes or macrophages.
  • the binding domains antagonize at least one activity of FcyRIIB.
  • said activity is activation of B cell receptor- mediated signaling.
  • the binding domains enhance B cell activity, B cell proliferation, antibody production, intracellular calcium influx, or activity of one or more downstream signaling molecules in the FcyRIIB signal transduction pathway.
  • the binding domains decrease phosphorylation of FcyRIIB or SHIP recruitment.
  • the binding domains enhance MAP kinase activity or Akt recruitment in the B cell receptor mediated signaling pathway.
  • the binding domains antagonize FcyRI IB -mediated inhibition of FceRI signaling.
  • the binding domains enhance FcsRI-induced mast cell activation, calcium mobilization, degranulation, cytokine production, or serotonin release.
  • the binding domains inhibit phosphorylation of FcyRIIB, inhibit recruitment of SHIP, inhibit SHIP phosphorylation and its association with She, enhance activation of MAP kinase family members (e.g. , Erkl , Erk2, JNK, p38, etc.).
  • the binding domains inhibit tyrosine phosphorylation of p62dok and its association with SHIP and rasGAP.
  • the binding domains enhance FcyR-mediated phagocytosis in monocytes or macrophages.
  • the binding domains prevent phagocytosis, clearance of opsonized particles by splenic macrophages.
  • at least one of the binding domains can be used to target the diabodies of the invention to cells that express FcyRIIB.
  • one of the binding domains is derived from a mouse monoclonal antibody produced by clone 2B6 or 3H7, having ATCC accession numbers PTA-4591 and PTA-4592, respectively.
  • Hybridomas producing antibodies 2B6 and 3H7 have been deposited with the American Type Culture Collection (10801
  • the binding domains are human or have been humanized, preferably are derived from a humanized version of the antibody produced by clone 3H7 or 2B6.
  • the invention also encompasses diabodies with binding domains from other antibodies, that specifically bind FcyRIIB, preferably human FcyRIIB, more preferably native human FcyRIIB, that are derived from clones including but not limited to 1 D5, 2E1 , 2H9, 2D 1 1 , and 1 F2 having ATCC Accession numbers, PTA-5958, PTA-5961 , PTA-5962, PTA-5960, and PTA-5959, respectively.
  • Hybridomas producing the above- identified clones were deposited under the provisions of the Budapest Treaty with the American Type Culture Collection (10801 University Boulevard., Manassas, VA. 201 10-2209) on May 7, 2004, and are incorporated herein by reference.
  • the binding domains from the antibodies described above are humanized.
  • the binding domains used in the diabodies of the present invention are from an antibody or an antigen-binding fragment thereof (e.g., comprising one or more complementarily determining regions (CDRs), preferably all 6 CDRs) of the antibody produced by clone 2B6, 3H7, 1 D5, 2E1 , 2H9, 2D 1 1 , or 1 F2.
  • CDRs complementarily determining regions
  • the binding domain binds to the same epitope as the mouse monoclonal antibody produced from clone 2B6, 3H7, 1 D5, 2E1 , 2H9, 2D 1 1 , or 1 F2, respectively and/or competes with the mouse monoclonal antibody produced from clone 2B6, 3H7, 1 D5, 2E1 , 2H9, 2D1 1 , or 1 F2 as determined, e.g., in an ELISA assay or other appropriate competitive immunoassay, and also binds FcyRIIB with a greater affinity than the binding domain binds FcyRIIA.
  • the present invention also encompasses diabodies with binding domains comprising an amino acid sequence of a variable heavy chain and/or variable light chain that is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of the variable heavy chain and/or light chain of the mouse monoclonal antibody produced by clone 2B6, 3H7, 1D5, 2E1, 2H9, 2D1 1, or 1F2.
  • the present invention further encompasses diabodies with binding domains that specifically bind FcyRIIB with greater affinity than said antibody or fragment thereof binds FcyRIIA, and that comprise an amino acid sequence of one or more CDRs that is at least 45%, at least 50%., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of one or more CDRs of the mouse monoclonal antibody produced by clone 2B6, 3H7, 1D5, 2E1 , 2H9, 2D1 1 , or 1 F2.
  • the determination of percent identity of two amino acid sequences can be determined by any method known to one skilled in the art, including BLAST protein searches.
  • the present invention also encompasses the use of diabodies containing binding domains that specifically bind FcyRIIB with greater affinity than binding domain binds FcyRIIA, which are encoded by a nucleotide sequence that hybridizes to the nucleotide sequence of the mouse monoclonal antibody produced by clone 2B6, 3H7, 1 D5, 2E1 , 2H9, 2D 1 1 , or 1 F2 under stringent conditions.
  • the binding domain specifically binds FcyRIIB with greater affinity than FcyRIIA, and comprises a variable light chain and/or variable heavy chain encoded by a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the variable light chain and/or variable heavy chain of the mouse monoclonal antibody produced by clone 2B6, 3H7, 1 D5, 2E1 , 2H9, 2D 1 1 , or 1 F2 under stringent conditions.
  • the binding domains specifically bind FcyRIIB with greater affinity than FcyRIIA, and comprise one or more CDRs encoded by a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of one or more CDRs of the mouse monoclonal antibody produced by clone 2B6, 3H7, 1 D5, 2E1 , 2H9, 2D 1 1 , or 1 F2.
  • Stringent hybridization conditions include, but are not limited to, hybridization to filter-bound DNA in 6X sodium chloride/sodium citrate (SSC) at about 45°C followed by one or more washes in 0.2X SSC/0.1 % SDS at about 50-65°C, highly stringent conditions such as hybridization to filter-bound DNA in 6X SSC at about 45°C followed by one or more washes in 0.1 X SSC/0.2% SDS at about 60°C, or any other stringent hybridization conditions known to those skilled in the art (see, for example, Ausubel, F.M. et al, eds. 1989 Current Protocols in Molecular Biology, vol. 1 , Green Publishing Associates, Inc. and John Wiley and Sons, Inc., NY at pages 6.3.1 to 6.3.6 and 2.10.3, incorporated herein by reference).
  • SSC sodium chloride/sodium citrate
  • the present invention also encompasses the use of binding domains comprising the amino acid sequence of any of the binding domains described above with mutations (e.g., one or more amino acid substitutions) in the framework or CDR regions.
  • mutations in these binding domains maintain or enhance the avidity and/or affinity of the binding domains for FcyRIIB to which they immunospecifically bind.
  • Standard techniques known to those skilled in the art can be used to assay the affinity of an antibody for a particular antigen.
  • Standard techniques known to those skilled in the art can be used to introduce mutations in the nucleotide sequence encoding an antibody, or fragment thereof, including, e.g. , site-directed mutagenesis and PCR-mediated mutagenesis, which results in amino acid substitutions.
  • the derivatives include less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the original antibody or fragment thereof.
  • the derivatives have conservative amino acid substitutions made at one or more predicted non-essential amino acid residues.
  • binding domains are derived from humanized antibodies.
  • a humanized FcyRIIB specific antibody may comprise
  • all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.
  • the diabodies of present invention comprise humanized variable domains specific for FcyRIIB in which one or more regions of one or more CDRs of the heavy and/or light chain variable regions of a human antibody (the recipient antibody) have been substituted by analogous parts of one or more CDRs of a donor monoclonal antibody which specifically binds FcyRIIB, with a greater affinity than FcyRIIA, e.g. , a monoclonal antibody produced by clone 2B6, 3H7, 1 D5, 2E1 , 2H9, 2D1 1 , or 1 F2.
  • the humanized antibodies bind to the same epitope as 2B6, 3H7, 1D5, 2E1, 2H9, 2D 1 1, or 1F2, respectively.
  • the CDR regions of the humanized FcyRIIB binding domain are derived from a murine antibody specific for FcyRIIB.
  • the humanized antibodies described herein comprise alterations, including but not limited to amino acid deletions, insertions, modifications, of the acceptor antibody, i.e., human, heavy and/or light chain variable domain framework regions that are necessary for retaining binding specificity of the donor monoclonal antibody.
  • the framework regions of the humanized antibodies described herein does not necessarily consist of the precise amino acid sequence of the framework region of a natural occurring human antibody variable region, but contains various alterations, including but not limited to amino acid deletions, insertions, modifications that alter the property of the humanized antibody, for example, improve the binding properties of a humanized antibody region that is specific for the same target as the murine FcyRIIB specific antibody.
  • a minimal number of alterations are made to the framework region in order to avoid large-scale introductions of non-human framework residues and to ensure minimal immunogenicity of the humanized antibody in humans.
  • the donor monoclonal antibody is preferably a monoclonal antibody produced by clones 2B6, 3H7, 1 D5, 2E1 , 2H9, 2Dl l , or 1 F2.
  • the binding domain encompasses variable domains of a CDR-grafted antibody which specifically binds FcyRIIB with a greater affinity than said antibody binds FcyRIIA, wherein the CDR-grafted antibody comprises a heavy chain variable region domain comprising framework residues of the recipient antibody and residues from the donor monoclonal antibody, which specifically binds FcyRIIB with a greater affinity than said antibody binds FcyRIIA, e.g. , monoclonal antibody produced from clones 2B6, 3H7, 1 D5, 2E1 , 2H9, 2D 1 1 , or 1 F2.
  • the diabodies of the invention comprise variable domains from a CDR-grafted antibody which specifically binds FcyRIIB with a greater affinity than said antibody binds FcyRIIA, wherein the CDR-grafted antibody comprises a light chain variable region domain comprising framework residues of the recipient antibody and residues from the donor monoclonal antibody, which specifically binds FcyRIIB with a greater affinity than said antibody binds FcyRIIA, e.g., monoclonal antibody produced from clones 2B6, 3H7, 1 D5, 2E1 , 2H9, 2Dl l, or 1F2.
  • the humanized anti- FcyRIIB variable domains used in the invention may have a heavy chain variable region comprising the amino acid sequence of CDR1 (SEQ ID NO: 24 or SEQ ID NO: 25) and/or CDR2 (SEQ ID NO: 26 or SEQ ID NO: 27) and/or CDR3 (SEQ ID NO: 28 or SEQ ID NO: 29) and/or a light chain variable region comprising the amino acid sequence of CDR1 (SEQ ID NO: 30 or SEQ ID NO: 31) and/or a CDR2 (SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 35) and/or CDR3 (SEQ ID NO: 36 or SEQ ID NO: 37).
  • the diabody comprises variable domains from a humanized 2B6 antibody, wherein the VH region consists of the FR segments from the human germline VH segment VH1-18 (Matsuda et al. (1998) "The Complete Nucleotide Sequence Of The Human Immunoglobulin Heavy Chain Variable Region Locus, " J. Exp. Med. 188:2151-2162) and JH6 (Ravetch et al. (1981 ) "Structure Of The Human
  • Immunoglobulin Mu Locus Characterization Of Embryonic And Rearranged J And D Genes, " Cell 27(3 Pt. 2): 583-91 ), and one or more CDR regions of the 2B6 VH, having the amino acid sequence of SED ID NO:24, SEQ ID NO: 26, or SEQ ID NO: 28.
  • the 2B6 VH has the amino acid sequence of SEQ ID NO: 38.
  • the 2B6 VH domain has the amino acid sequence of Hu2B6VH, SEQ ID NO: 85, and can be encoded by the nucleotide sequence of SEQ ID NO: 86.
  • the diabody further comprises a VL region, which consists of the FR segments of the human germline VL segment V -A26 (Lautner-Rieske et al. (1992) "The Human Immunoglobulin Kappa Locus. Characterization Of The Duplicated A Regions, " Eur. J. Immunol. 22: 1023-1029) and JK4 (Hieter et al. (1982) "Evolution Of Human Immunoglobulin Kappa J Region Genes, " J. Biol. Chem.
  • the 2B6 VL has the amino acid sequence of SEQ ID NO: 39; SEQ ID NO: 40, or SEQ ID NO: 41.
  • the 2B6 VL has the amino acid sequence of Hu2B6VL, SEQ ID NO: 87, and can be encoded by the nucleotide sequence provided in SEQ ID NO: 88.
  • the diabody has variable domains from a humanized 3H7 antibody, wherein the VH region consists of the FR segments from a human germline VH segment and the CDR regions of the 3H7 VH, having the amino acid sequence of SEQ ID NO. 35.
  • the humanized 3H7 antibody further comprises a VL regions, which consists of the FR segments of a human germline VL segment and the CDR regions of 3H7VL, having the amino acid sequence of SEQ ID NO: 42.
  • binding domains immunospecifically bind to extracellular domains of native human FcyRIIB, and comprise (or alternatively, consist of) CDR sequences of 2B6, 3H7, 1D5, 2E1 , 2H9, 2D 1 1 , or 1 F2, in any of the following
  • VH CDRl and a VL CDRl combinations: a VH CDRl and a VL CDRl ; a VH CDRl and a VL CDR2; a VH CDRl and a VL CDR3; a VH CDR2 and a VL CDRl ; VH CDR2 and VL CDR2; a VH CDR2 and a VL CDR3; a VH CDR3 and a VH CDRl ; a VH CDR3 and a VL CDR2; a VH CDR3 and a VL CDR3; a VH1 CDRl , a VH CDR2 and a VL CDRl ; a VH CDRl , a VH CDR2 and a VL CDR2; a VH CDRl , a VH CDR2 and a VL CDR3; a VH CDR2, a VH CDR3 and a VL
  • Antibodies for deriving binding domains to be included in the diabodies of the invention may be further characterized by epitope mapping, so that antibodies may be selected that have the greatest specificity for FcyRIIB compared to FeyRIIA.
  • Epitope mapping methods of antibodies are well known in the art and encompassed within the methods of the invention.
  • fusion proteins comprising one or more regions of FcyRIIB may be used in mapping the epitope of an antibody of the invention.
  • the fusion protein contains the amino acid sequence of a region of an FcyRIIB fused to the Fc portion of human IgG2.
  • Each fusion protein may further comprise amino acid substitutions and/or replacements of certain regions of the receptor with the corresponding region from a homolog receptor, e.g., FcyRIIA, as shown in Table 2 below.
  • FcyRIIA a homolog receptor
  • pMGX125 and pMGX132 contain the IgG binding site of the FcyRIIB receptor, the former with the C-terminus of FcyRIIB and the latter with the C-terminus of FcyRIIA and can be used to differentiate C-terminus binding.
  • the others have FcyRIIA
  • substitutions in the IgG binding site and either the FcyllA or FcyllB N-terminus can help determine the part of the receptor molecule where the antibodies bind.
  • Residues 172 to 180 belong to the IgG binding site of FcyRIIA and B.
  • FcyRIIA sequence are in bold.
  • the fusion proteins may be used in any biochemical assay for
  • the antibodies can be characterized using assays for identifying the function of the antibodies of the invention, particularly the activity to modulate FcyRIIB signaling. For example, characterization assays of the invention can measure
  • the characterization assays of the invention can be cell-based or cell-free assays.
  • FcyRIIB is rapidly phosphorylated on tyrosine in its ITIM motif, and then recruits Src Homology-2 containing inositol-5-phosphatase (SHIP), an SH2 domain-containing inosital polyphosphate 5-phosphatase, which is in turn phosphorylated and associates with She and p62 dok
  • SHIP inositol-5-phosphatase
  • p62 dok is the prototype of a family of adaptor molecules, which includes signaling domains such as an aminoterminal pleckstrin homology domain (PH domain), a PTB domain, and a carboxy terminal region containing PXXP motifs and numerous phosphorylation sites (Carpino et al.
  • the anti-FcyRIIB antibodies for use in the invention may likewise be characterized for ability to modulate one or more IgE mediated responses.
  • cells lines co-expressing the high affinity receptor for IgE and the low affinity receptor for FcyRIIB will be used in characterizing the anti-FcyRIIB antibodies in modulating IgE mediated responses.
  • cells from a rat basophilic leukemia cell line RBL-H23; Barsumian E.L. el al. (1981 ) "IgE-Induced Histamine Release From Rat Basophilic Leukemia Cell Lines: Isolation Of Releasing And Nonreleasing Clones, " Eur. J.
  • Immunol.1 1 :317-323 which is incorporated herein by reference in its entirety
  • transfected with full length human FcyRIIB will be used.
  • RBL-2H3 is a well characterized rat cell line that has been used extensively to study the signaling
  • FcyRIIB inhibits FceRI-induced calcium mobilization, degranulation, and cytokine production
  • FcyRIIB inhibits FceRI-induced calcium mobilization, degranulation, and cytokine production
  • Antibodies for use in the invention may also be characterized for inhibition of FceRI induced mast cell activation.
  • cells from a rat basophilic leukemia cell line RBL-H23; Barsumian E.L. et al. (1981) "IgE-Induced Histamine Release From Rat Basophilic Leukemia Cell Lines: Isolation Of Releasing And Nonreleasing Clones, " Eur. J.
  • Immunol.1 1 :317-323) that have been transfected with FcyRIIB are sensitized with IgE and stimulated either with F(ab') 2 fragments of rabbit anti-mouse IgG, to aggregate FceRI alone, or with whole rabbit anti-mouse IgG to coaggregate FcyRIIB and FceRI.
  • indirect modulation of down stream signaling molecules can be assayed upon addition of antibodies of the invention to the sensitized and stimulated cells.
  • tyrosine phosphorylation of FcyRIIB and recruitment and phosphorylation of SHIP activation of MAP kinase family members, including but not limited to Erkl , Erk2, J K, or p38; and tyrosine phosphorylation of p62 dok and its association with SHIP and RasGAP can be assayed.
  • One exemplary assay for determining the inhibition of FceRI induced mast cell activation by the antibodies of the invention can comprise of the following:
  • transfecting RBL-H23 cells with human FcyRIIB sensitizing the RBL-H23 cells with IgE; stimulating RBL-H23 cells with either F(ab') 2 of rabbit anti-mouse IgG (to aggregate FceRI alone and elicit FceRI-mediated signaling, as a control), or stimulating RBL-H23 cells with whole rabbit anti-mouse IgG to (to coaggregate FcyRIIB and FceRI, resulting in inhibition of FceRI-mediated signaling).
  • Cells that have been stimulated with whole rabbit anti-mouse IgG antibodies can be further pre-incubated with the antibodies of the invention.
  • the exemplary assay described above can be for example, used to identify antibodies that block ligand (IgG) binding to FcyRIIB receptor and antagonize FcyRIIB- mediated inhibition of FceRI signaling by preventing coaggregating of FcyRIIB and FceRI.
  • This assay likewise identifies antibodies that enhance coaggregation of FcyRIIB and FceRI and agonize FcyRIIB-mediated inhibition of FceRI signaling by promoting coaggregating of FcyRIIB and FceRI.
  • the anti-FcyRIIB diabodies comprising the epitope binding domains of anti-FcyRIIB antibodies identified described herein or known in the art, of the invention are characterized for their ability to modulate an IgE mediated response by monitoring and/or measuring degranulation of mast cells or basophils, preferably in a cell-based assay.
  • mast cells or basophils for use in such assays have been engineered to contain human FcyRIIB using standard recombinant methods known to one skilled in the art.
  • the anti-FcyRIIB antibodies of the invention are characterized for their ability to modulate an IgE mediated response in a cell-based ⁇ -hexosaminidase (enzyme contained in the granules) release assay, ⁇ - hexosaminidase release from mast cells and basophils is a primary event in acute allergic and inflammatory condition (Aketani et al. (2001) "Correlation Between Cytosolic Calcium Concentration And Degranulation In RBL-2H3 Cells In The Presence Of Various Concentrations Of Antigen-Specific IgEs, " Immunol. Lett. 75: 185-189; Aketani et al.
  • LAD 1 and LAD2 novel stem cell factor dependent human mast cell lines
  • LAD 1 and 2 cells can be used for assessing the effect of the antibodies of the invention on IgE mediated responses.
  • cell-based ⁇ -hexosaminidase release assays such as those described supra may be used in LAD cells to determine any modulation of the IgE- mediated response by the anti-FcyRIIB antibodies of the invention.
  • human mast cells e.g., LAD 1
  • LAD 1 chimeric human IgE anti- nitrophenol (NP)
  • BSA-NP nitrophenol
  • the polyvalent antigen e.g., NP
  • cell degranulation is monitored by measuring the ⁇ -hexosaminidase released in the supernatant (Kirshenbaum et al. (2003) "Characterization Of Novel Stem Cell Factor Responsive Human Mast Cell Lines LAD I And 2 Established From A Patient With Mast Cell Sarcoma/Leukemia; Activation Following Aggregation Of FcRI Or FcyRI, " Leukemia research, 27:677-82, which is incorporated herein by reference in its entirety.).
  • FcyRIIB has been described to be highly up-regulated in human monocyte cell lines, e.g., THP1 and U937, (Tridandapani et al.
  • the anti-FcyRIIB diabodies can also be assayed for inhibition of B-cell receptor (BCR)-mediated signaling.
  • BCR-mediated signaling can include at least one or more down stream biological responses, such as activation and proliferation of B cells, antibody production, etc.
  • Coaggregation of FcyRIIB and BCR leads to inhibition of cell cycle progression and cellular survival. Further, coaggregation of FcyRIIB and BCR leads to inhibition of BCR-mediated signaling.
  • BCR-mediated signaling comprises at least one or more of the following: modulation of down stream signaling molecules (e.g., phosphorylation state of FcyRIIB, SHIP recruitment, localization of Btk and/or PLCy, MAP kinase activity, recruitment of Akt (anti-apoptotic signal), calcium mobilization, cell cycle progression, and cell proliferation.
  • modulation of down stream signaling molecules e.g., phosphorylation state of FcyRIIB, SHIP recruitment, localization of Btk and/or PLCy
  • MAP kinase activity e.g., MAP kinase activity
  • Akt anti-apoptotic signal
  • calcium mobilization e.g., calcium mobilization, cell cycle progression, and cell proliferation.
  • ex vivo B cells from SHIP deficient mice can be used to characterize the antibodies of the invention.
  • One exemplary assay for determining FcyRIIB-mediated inhibition of BCR signaling by the antibodies of the invention can comprise the following: isolating splenic B cells from SHIP deficient mice, activating said cells with lipopolysachharide, and stimulating said cells with either F(ab') 2 anti-lgM to aggregate BCR or with anti-lgM to coaagregate BCR with FcyRIIB.
  • FcyRIIB-dependent activity of cells can be measured by standard techniques known in the art. Comparing the level of FcyRIIB-dependent activity in cells that have been pre-incubated with the antibodies and cells that have not been pre-incubated, and comparing the levels would indicate a modulation of FcyRIIB-dependent activity by the antibodies.
  • Measuring FcyRIIB-dependent activity can include, for example, measuring intracellular calcium mobilization by flow cytometry, measuring phosphorylation of Akt and/or Erk, measuring BCR-mediated accumulation of PI(3,4,5)P 3 , or measuring FcyRIIB-mediated proliferation B cells.
  • the assays can be used, for example, to identify diabodies or anti-FcyRIIB antibodies for use in the invention that modulate FcyRIIB-mediated inhibition of BCR signaling by blocking the ligand (IgG) binding site to FcyRIIB receptor and antagonizing FcyRIIB-mediated inhibition of BCR signaling by preventing coaggregation of FcyRIIB and BCR.
  • the assays can also be used to identify antibodies that enhance coaggregation of FcyRIIB and BCR and agonize FcyRIIB-mediated inhibition of BCR signaling.
  • the anti-FcyRIIB antibodies can also be assayed for FcyRII-mediated signaling in human monocytes/macrophages.
  • Coaggregation of FcyRIIB with a receptor bearing the immunoreceptor tyrosine-based activation motif (ITAM) acts to down-regulate FcyR-mediated phagocytosis using SHIP as its effector (Tridandapani et al. (2002) "Regulated Expression And Inhibitory Function Of Fcgamma RUB In Human Monocytic Cells, " J. Biol. Chem., 277(7): 5082-5089).
  • Coaggregation of FcyRIIA with FcyRIIB results in rapid phosphorylation of the tyrosine residue on FcyRIIB 's ITIM motif, leading to an enhancement in phosphorylation of SHIP, association of SHIP with She, and phosphorylation of proteins having the molecular weight of 120 and 60-65 kDa.
  • coaggregation of FcyRIIA with FcyRIIB results in down-regulation of phosphorylation of Akt, which is a serine-threonine kinase that is involved in cellular regulation and serves to suppress apoptosis.
  • the anti-FcyRIIB diabodies can also be assayed for inhibition of FcyR- mediated phagocytosis in human monocytes/macrophages.
  • THP-1 can be stimulated either with Fab fragments of mouse monoclonal antibody IV.3 against FcyRII and goat anti-mouse antibody (to aggregate FcyRIIA alone), or with whole IV.3 mouse monoclonal antibody and goat anti-mouse antibody (to coaggregate FcyRIIA and FcyRIIB).
  • modulation of down stream signaling molecules such as tyrosine phosphorylation of FcyRIIB, phosphorylation of SHIP, association of SHIP with She, phosphorylation of Akt, and phosphorylation of proteins having the molecular weight of 120 and 60-65 kDa can be assayed upon addition of molecules of the invention to the stimulated cells.
  • FcyRIIB-dependent phagocytic efficiency of the monocyte cell line can be directly measured in the presence and absence of the antibodies of the invention.
  • Another exemplary assay for determining inhibition of FcyR-mediated phagocytosis in human monocytes/macrophages by the antibodies of the invention can comprise the following: stimulating THP-1 cells with either Fab of IV.3 mouse anti- FcyRII antibody and goat anti-mouse antibody (to aggregate FcyRIIA alone and elicit FcyRIIA-mediated signaling); or with mouse anti-FcyRII antibody and goat anti-mouse antibody (to coaggregate FcyRIIA and FcyRIIB and inhibiting FcyRIIA-mediated signaling.
  • Cells that have been stimulated with mouse anti-FcyRII antibody and goat anti- mouse antibody can be further pre-incubated with the molecules of the invention.
  • FcyRIIA-dependent activity of stimulated cells that have been pre-incubated with molecules of the invention and cells that have not been pre-incubated with the antibodies of the invention and comparing levels of FcyRIIA-dependent activity in these cells would indicate a modulation of FcyRIIA-dependent activity by the antibodies of the invention.
  • the exemplary assay described can be used for example, to identify binding domains that block ligand binding of FcyRIIB receptor and antagonize FcyRIIB- mediated inhibition of FcyRIIA signaling by preventing coaggregation of FcyRIIB and FcyRIIA.
  • This assay likewise identifies binding domains that enhance coaggregation of FcyRIIB and FcyRIIA and agonize FcyRIIB-mediated inhibition of FcyRIIA signaling.
  • the FcyRIIB binding domains of interest can be assayed while comprised I antibodies by measuring the ability of THP-1 cells to phagocytose fluoresceinated IgG- opsonized sheep red blood cells (SRBC) by methods previously described (Tridandapani et al. (2000) "The Adapter Protein LAT Enhances Fcgamma Receptor-Mediated Signal Transduction In Myeloid Cells, " J. Biol. Chem. 275: 20480-7).
  • SRBC phagocytose fluoresceinated IgG- opsonized sheep red blood cells
  • an exemplary assay for measuring phagocytosis comprises of: treating THP-1 cells with the antibodies of the invention or with a control antibody that does not bind to FcyRII, comparing the activity levels of said cells, wherein a difference in the activities of the cells (e.g. , rosetting activity (the number of THP-1 cells binding IgG-coated SRBC), adherence activity (the total number of SRBC bound to THP-1 cells), and phagocytic rate) would indicate a modulation of FcyRIIA-dependent activity by the antibodies of the invention.
  • a difference in the activities of the cells e.g. , rosetting activity (the number of THP-1 cells binding IgG-coated SRBC), adherence activity (the total number of SRBC bound to THP-1 cells), and phagocytic rate
  • This assay can be used to identify, for example, antibodies that block ligand binding of FcyRIIB receptor and antagonize FcyRIIB-mediated inhibition of phagocytosis. This assay can also identify antibodies that enhance FcyRIIB-mediated inhibition of FcyRIIA signaling.
  • the binding domains modulate FcyRIIB- dependent activity in human monocytes/macrophages in at least one or more of the following ways: modulation of downstream signaling molecules (e.g., modulation of phosphorylation state of FcyRIIB, modulation of SHIP phosphorylation, modulation of SHIP and She association, modulation of phosphorylation of Akt, modulation of phosphorylation of additional proteins around 120 and 60-65 kDa) and modulation of phagocytosis.
  • downstream signaling molecules e.g., modulation of phosphorylation state of FcyRIIB, modulation of SHIP phosphorylation, modulation of SHIP and She association, modulation of phosphorylation of Akt, modulation of phosphorylation of additional proteins around 120 and 60-65 kDa
  • CD16A binding proteins which can be used as sources for light and heavy chain variable regions for covalent diabody
  • CD16A binding proteins includes molecules comprising VL and VH domains of anti-CD 16A antibodies, which VH and VL domains are used in the production of the diabodies of the present invention.
  • CD16A binding proteins include human or humanized monoclonal antibodies as well as CD16A binding antibody fragments (e.g., scFv or single chain antibodies, Fab fragments, minibodies) and another antibody-like proteins that bind to CD16A via an interaction with a light chain variable region domain, a heavy chain variable region domain, or both.
  • CD16A binding antibody fragments e.g., scFv or single chain antibodies, Fab fragments, minibodies
  • another antibody-like proteins that bind to CD16A via an interaction with a light chain variable region domain, a heavy chain variable region domain, or both.
  • the CD16A binding protein for use according to the invention comprises a VL and/or VH domain that has one or more CDRs with sequences derived from a non-human anti-CD 16A antibody, such as a mouse anti-CD 16A antibody, and one or more framework regions with derived from framework sequences of one or more human immunoglobulins.
  • a non-human anti-CD 16A antibody such as a mouse anti-CD 16A antibody
  • framework regions with derived from framework sequences of one or more human immunoglobulins.
  • a number of non-human anti-CD 16A monoclonal antibodies, from which CDR and other sequences may be obtained, are known (see, e.g., Tamm et al. (1996) "The Binding Epitopes Of Human CD 16 (Fc gamma Rill)
  • new CD16A binding proteins that recognize human CD16A expressed on cells can be obtained using well known methods for production and selection of monoclonal antibodies or related binding proteins (e.g., hybridoma technology, phage display, and the like). See, for example, O'Connell et al. (2002) "Phage Versus Phagemid Libraries For Generation Of Human Monoclonal Antibodies, " J. Mol. Biol. 321 :49-56; Hoogenboom et al (2000) "Natural And Designer Binding Sites Made By Phage Display Technology, " Imm. Today 21 :371078; Krebs et al. (2001) "High-Throughput Generation And Engineering Of Recombinant Human Antibodies, " J. Imm. Methods 254:67-84; and other references cited herein. Monoclonal antibodies from a non-human species can be chimerized or humanized using techniques using techniques of antibody humanization known in the art.
  • Fully human antibodies against CD16A can be produced using transgenic animals having elements of a human immune system (see, e.g., U.S. Pat. Nos. 5,569,825 and 5,545,806), using human peripheral blood cells (Casali et al. (1986) "Human Monoclonals From Antigen-Specific Selection Of B Lymphocytes And
  • the binding donor is from the 3G8 antibody or a humanized version thereof, e.g., such as those disclosed in U.S. patent application publication 2004/0010124, which is incorporated by reference herein in its entirety. It is contemplated that, for some purposes, it may be advantageous to use CD16A binding proteins that bind the CD16A receptor at the same epitope bound by 3G8, or at least sufficiently close to this epitope to block binding by 3G8. Methods for epitope mapping and competitive binding experiments to identify binding proteins with the desired binding properties are well known to those skilled in the art of experimental immunology. See, for example, Harlow and Lane, cited supra; Stahli et al.
  • Epitope comparison can also be achieved by labeling a first antibody, directly or indirectly, with an enzyme, radionuclide or fluorophore, and measuring the ability of an unlabeled second antibody to inhibit the binding of the first antibody to the antigen on cells, in solution, or on a solid phase.
  • immune complexes formed on ELISA plates.
  • Such immune complexes are formed by first coating the plate with an antigen such as fluorescein, then applying a specific anti-fluorescein antibody to the plate.
  • This immune complex serves as the ligand for soluble Fc receptors such as sFcRIIIa.
  • a soluble immune complex may be formed and labeled, directly or indirectly, with an enzyme radionuclide or fluorophore.
  • the ability of antibodies to inhibit the binding of these labeled immune complexes to Fc receptors on cells, in solution or on a solid phase can then be measured.
  • CD16A binding proteins of the invention may or may not comprise a human immunoglobulin Fc region. Fc regions are not present, for example, in scFv binding proteins. Fc regions are present, for example, in human or humanized tetrameric monoclonal IgG antibodies. As described supra, in some embodiments of the present invention, the CD16A binding protein includes an Fc region that has an altered effector function, e.g., reduced affinity for an effector ligand such as an Fc receptor or CI component of complement compared to the unaltered Fc region (e.g. , Fc of naturally occurring IgGl , proteins). In one embodiment the Fc region is not glycosylated at the Fc region amino acid corresponding to position 297. Such antibodies lack Fc effector function.
  • an effector function e.g., reduced affinity for an effector ligand such as an Fc receptor or CI component of complement compared to the unaltered Fc region (e.g. , Fc of naturally occurring Ig
  • the CD16A binding protein may not exhibit Fc-mediated binding to an effector ligand such as an Fc receptor or the C I component of complement due to the absence of the Fc domain in the binding protein while, in other cases, the lack of binding or effector function is due to an alteration in the constant region of the antibody.
  • an effector ligand such as an Fc receptor or the C I component of complement due to the absence of the Fc domain in the binding protein while, in other cases, the lack of binding or effector function is due to an alteration in the constant region of the antibody.
  • CD16A binding proteins that can be used in the practice of the invention include proteins comprising a CDR sequence derived from (i.e., having a sequence the same as or similar to) the CDRs of the mouse monoclonal antibody 3G8.
  • the nucleic acid and protein sequences of 3G8 are provided below. Using the mouse variable region and CDR sequences, a large number of chimeric and humanized monoclonal antibodies, comprising complementary determining regions derived from 3G8
  • CDRs were produced and their properties analyzed.
  • antibody heavy chains comprising a VH region with CDRs derived from 3G8 were produced and combined (by coexpression) with antibody light chains comprising a VL region with CDRs derived from 3G8
  • CD16A binding proteins comprising 3G8 CDRs, such as the humanized antibody proteins described herein, may be used according to the invention.
  • the CD16A binding protein of the invention may comprise a heavy chain variable domain in which at least one CDR (and usually three CDRS) have the sequence of a CDR (and more typically all three CDRS) of the mouse monoclonal antibody 3G8 heavy chain and for which the remaining portions of the binding protein are substantially human (derived from and substantially similar to, the heavy chain variable region of a human antibody or antibodies).
  • the invention provides a humanized 3G8 antibody or antibody fragment containing CDRs derived from the 3G8 antibody in a substantially human framework, but in which at least one of the CDRs of the heavy chain variable domain differs in sequence from the corresponding mouse antibody 3G8 heavy chain CDR.
  • the CDR(S) differs from the 3G8 CDR sequence at least by having one or more CDR substitutions shown known in the art to affect binding of 3G8 to CD16A, as known in the art or as disclosed in Tables 3 and 4A-H.
  • Suitable CD 16 binding proteins may comprise 0, 1, 2, 3, or 4, or more of these substitutions (and often have from 1 to 4 of these substitutions) and optionally can have additional substitutions as well.
  • a CD16A binding protein may comprise a heavy chain variable domain sequence that is the same as, or similar to, the VH domain of the
  • the invention provides a CD16A binding protein comprising a VH domain with a sequence that (1) differs from the VH domain of Hu3G8VH-l (SEQ ID NO: 68) by zero, one, or more than one of the CDR substitutions set forth in Table 1 ; (2) differs from the VH domain of Hu3G8VH- l by zero, one or more than one of the framework substitutions set forth in Table 1; and (3) is at least about 80% identical, often at least about 90%, and sometimes at least about 95% identical, or even at least about 98% identical to the
  • Exemplary VH domains of CD 16 binding proteins of the invention have the sequence of 3G8VH, Hu3G8VH-5 and Hu3G8VH-22 (SEQ ID NO: 79, SEQ ID NO: 69 and SEQ ID NO: 70, respectively).
  • Examplary nucleotide sequences encoding the sequences of 3G8VH and Hu3G8VH-5 are provided by SEQ ID NO: 80 and SEQ ID NO: 81, respectively.
  • the VH domain may have a sequence that differs from that of Hu3G8VH-l
  • the degree of sequence identity with the Hu3G8VH-l VH domain at the remaining positions is at least about 80%, at least about 90%, at least about 95% or at least about 98%.
  • CD16A building protein VH domains For illustration and not limitation, the sequences of a number of CD16A building protein VH domains is shown in Table 4. Heavy chains comprising these sequences fused to a human Cyl constant region were coexpressed with the hu3G8VL-l light chain (described below) to form tetrameric antibodies, and binding of the antibodies to CD16A was measured to assess the effect of amino acid substitutions compared to the hu3G8VH-l VH domain. Constructs in which the VH domain has a sequence of hu3G8VH-l , 2, 3, 4, 5, 8, 12, 14. 16, 17, 18, 19, 20, 22.
  • CD16A binding proteins comprising the VH domains of hu3G8VH-5 and hu3G8VH-22 (SEQ ID NO: 69 and SEQ ID NO: 70, respectively) are considered to have particularly favorable binding properties.
  • the invention provides a CD16A binding protein containing a light chain variable domain in which at least one CDR (and usually three CDRs) has the sequence of a CDR (and more typically all three CDRs) of the mouse monoclonal antibody 3G8 light chain and for which the remaining portions of the binding protein are substantially human (derived from and substantially similar to, the heavy chain variable region of a human antibody or antibodies).
  • the invention provides a fragment of a humanized 3G8 antibody containing CDRs derived from the 3G8 antibody in a substantially human framework, but in which at least one of the CDRs of the light chain variable domain differs in sequence from the mouse monoclonal antibody 3G8 light chain CDR.
  • the CDR(s) differs from the 3G8 sequence at least by having one or more amino acid substitutions in a CDR, such as, one or more substitutions shown in Table 2 (e.g., arginine at position 24 in CDRl ; serine at position 25 in CDRl ; tyrosine at position 32 in CDRl ; leucine at position 33 in CDRl ; aspartic acid, tryptophan or serine at position 50 in CDR2; serine at position 53 in CDR2; alanine or glutamine at position 55 in CDR2; threonine at position 56 in CDR2; serine at position 93 in CDR3; and/or threonine at position 94 in CDR3).
  • the variable domain can have 0, 1 , 2, 3, 4,
  • substitutions 1, or more of these substitutions (and often have from 1 to 4 of these substitutions) and optionally, can have additional substitutions as well.
  • a suitable CD16A binding protein may comprise a light chain variable domain sequence that is the same as, or similar to, the VL domain of the Hu3G8VL-l (SEQ ID NO: 71) construct, the sequence of which is provided in Table
  • the invention provides a CD16A binding protein comprising a VL domain with a sequence that (1) differs from the VL domain of Hu3G8VL-l (SEQ ID NO: 71) by zero, one, or more of the CDR substitutions set forth in Table 5; (2) differs from the VL domain of Hu3G8VL- l by zero, one or more of the framework substitutions set forth in Table 5; and (3) is at least about 80% identical, often at least about 90%, and sometimes at least about 95% identical, or even at least about 98% identical to the Hu3G8VL-l VL sequence (SEQ ID NO: 71) at the remaining positions.
  • Exemplary VL domains of CD 16 binding proteins of the invention have the sequence of 3G8VL, Hu3G8VL-l or Hu3G8VL-43, (SEQ ID NO: 82, SEQ ID NO: 71 and SEQ ID NO: 72, respectively) as shown in Tables 5 and 6.
  • Exemplary nucleotide sequences encoding 3G8VL (SEQ ID NO: 82) and Hu3G8VL-l (SEQ ID NO: 71) are provided in SEQ ID NO: 83 and SEQ ID NO: 84, respectively.
  • the VL domain may have a sequence that differs from that of Hu3G8VL- 1 (SEQ ID NO: 71) by zero, one, at least two, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 of the substitutions shown in Table 2. These substitutions are believed to result in increased affinity for CD16A and/or reduce the immunogenicity of a CD16A binding protein when administered to humans.
  • the degree of sequence identity at the remaining positions is at least about 80%, at least about 90% at least about 95% or at least about 98%.
  • VL domain has a sequence of hu3G8VL-l, 2, 3, 4, 5, 10, 16, 18, 19, 21 , 22, 24, 27, 28, 32, 33, 34, 35, 36, 37, and 42 showed high affinity binding and hu3G8VL-15, 17, 20, 23, 25, 26, 29, 30, 31 , 38, 39, 40 and 41 showed intermediate binding.
  • CD16A binding proteins comprising the VL domains of hu3G8VL-l , hu3G8VL-22, and hu3G8VL-43 are considered to have particularly favorable binding properties (SEQ ID NO: 71, SEQ ID NO: 73 and SEQ ID NO: 72, respectively).
  • immunoglobulin light and heavy chains can be recombinantly expressed under conditions in which they associate to produce a diabody, or can be so combined in vitro. It will thus be appreciated that a 3G8-derived VL-domain described herein can be combined a 3G8-derived VH- domain described herein to produce a CD16A binding diabody, and all such combinations are contemplated.
  • examples of useful CD16A diabodies are those comprising at least one VH domain and at least one VL domain, where the VH domain is from hu3G8VH-l , hu3G8VH-22 or hu3G8VH-5 (SEQ ID NO: 68, SEQ ID NO: 70 and SEQ ID NO: 69, respectively) and the VL domain is from hu3G8VL-l , hu3G8VL-22 or hu3G8VL-43 (SEQ ID NO: 71 , SEQ ID NO: 73 and SEQ ID NO: 41 , respectively).
  • humanized antibodies that comprise hu3G8VH-22 (SEQ ID NO: 22) and either, hu3G8VL-l , hu3G8VL-22 or hu3G8VL-43 (SEQ ID NO: 71 , SEQ ID NO: 70 and SEQ ID NO: 72, respectively), or hu3G8VH-5 (SEQ ID NO: 69) and hu3G8VL-l (SEQ ID NO: 71) have favorable properties.
  • CD16A binding proteins can be modified using affinity maturation techniques to identify proteins with increased affinity for CD16A and/or decreased affinity for CD16B.
  • CD 16 binding protein is the mouse 3G8 antibody.
  • Amino acid sequence comprising the VH and VL domains of humanized 3G8 are described in FIGS. 2, 9, 14 and set forth in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72.
  • the invention encompasses diabody molecules comprising Fc domains or portions thereof (e.g. , a CH2 or CH3 domain).
  • the Fc domain, or portion(s) thereof comprises one or more constant domain(s) of the Fc region of IgG2, IgG3 or IgG4 (e.g. , CH2 or CH3).
  • the invention encompasses molecules comprising and Fc domain or portion therof, wherein said Fc domain or portion thereof comprises at least one amino acid modification (e.g. substitution) relative to a comparable wild-type Fc domain or portion thereof.
  • Variant Fc domains are well known in the art, and are primarily used to alter the phenotype of the antibody comprising said variant Fc domain as assayed in any of the binding activity or effector function assays well known in the art, e.g. ELISA, SPR analysis, or ADCC.
  • Such variant Fc domains, or portions thereof have use in the present invention by conferring or modifying the effector function exhibited by a diabody molecule of the invention comprising an Fc domain (or portion thereof) as functionally assayed, e.g., in an N dependent or macrophage dependent assay.
  • Fc domain variants identified as altering effector function are disclosed in International Application WO04/063351 , U.S. Patent Application Publications
  • the invention encompasses the use of any Fc variant known in the art, such as those disclosed in Duncan et al. (1 88) "Localization Of The Binding Site For The Human High-Affinity Fc Receptor On IgG, " Nature 332:563-564; Lund et al. (1991) "Human Fc Gamma RI And Fc Gamma RII Interact With Distinct But Overlapping Sites On Human IgG, " J. Immunol. 147:2657-2662; Lund et al. (1992) “Multiple Binding Sites On The CHI Domain Of IgG For Mouse Fc Gamma RII, " Mol. Immunol.
  • said one or more modifications to the amino acids of the Fc region reduce the affinity and avidity of the Fc region and, thus, the diabody molecule of the invention, for one or more FcyR receptors.
  • the invention encompasses diabodies comprising a variant Fc region, or portion thereof, wherein said variant Fc region comprises at least one amino acid modification relative to a wild type Fc region, which variant Fc region only binds one FcyR, wherein said FcyR is FcyRIIIA.
  • the invention encompasses diabodies comprising a variant Fc region, or portion thereof, wherein said variant Fc region comprises at least one amino acid modification relative to a wild type Fc region, which variant Fc region only binds one FcyR, wherein said FcyR is FcyRIIA.
  • the invention encompasses diabodies comprising a variant Fc region, or portion thereof, wherein said variant Fc region comprises at least one amino acid modification relative to a wild type Fc region, which variant Fc region only binds one FcyR, wherein said FcyR is FcyRIIB.
  • the invention encompasses molecules comprising a variant Fc domain wherein said variant confers or mediates increased ADCC activity and/or an increased binding to FcyRIIA (CD32A), relative to a molecule comprising no Fc domain or comprising a wild-type Fc domain, as measured using methods known to one skilled in the art and described herein.
  • the invention encompasses molecules comprising a variant Fc domain wherein said variant confers or mediates decreased ADCC activity (or other effector function) and/or an increased binding to FcyRIIB (CD32B), relative to a molecule comprising no Fc domain or comprising a wild-type Fc domain, as measured using methods known to one skilled in the art and described herein.
  • the invention also encompasses the use of an Fc domain comprising domains or regions from two or more IgG isotypes.
  • amino acid modification of the Fc region can profoundly affect Fc-mediated effector function and/or binding activity.
  • these alterations in functional characteristics can be further refined and/or manipulated when implemented in the context of selected IgG isotypes.
  • the native characteristics of the isotype Fc may be manipulated by the one or more amino acid modifications.
  • the multiple IgG isotypes exhibit differing physical and functional properties including serum half-life, complement fixation, FcyR binding affinities and effector function activities (e.g. ADCC, CDC) due to differences in the amino acid sequences of their hinge and/or Fc domains.
  • the amino acid modification and IgG Fc region are independently selected based on their respective, separate binding and/or effector function activities in order to engineer a diabody with desired characteristics.
  • said amino acid modifications and IgG hinge/Fc regions have been separately assayed for binding and/or effector function activity as described herein or known in the art in an the context of an IgGl .
  • said amino acid modification and IgG hinge/Fc region display similar functionality, e.g. , increased affinity for FcyRIIA, when separately assayed for FcyR binding or effector function in the context of the diabody molecule or other Fc-containing molecule (e.g. and immunoglobulin).
  • amino acid modification and selected IgG Fc region then act additively or, more preferably, synergistically to modify said functionality in the diabody molecule of the invention, relative to a diabody molecule of the invention comprising a wild-type Fc region.
  • said amino acid modification and IgG Fc region display opposite functionalities, e.g., increased and decreased, respectively, affinity for FcyRIIA, when separately assayed for FcyR binding and/or effector function in the context of the diabody molecule or other Fc containing molecule (e.g.
  • an immunoglobulin comprising a wild- type Fc region as described herein or known in the art; the combination of said "opposite" amino acid modification and selected IgG region then act to selectively temper or reduce a specific functionality in the diabody of the invention relative to a diabody of the invention not comprising an Fc region or comprising a wild-type Fc region of the same isotype.
  • the invention encompasses variant Fc regions comprising combinations of amino acid modifications known in the art and selected IgG regions that exhibit novel properties, which properties were not detectable when said modifications and/or regions were independently assayed as described herein. [00239]
  • the functional characteristics of the multiple IgG isotypes, and domains thereof, are well known in the art.
  • IgG 1 , IgG2, IgG3 and IgG4 are presented in FIGS. 1A-1B. Selection and/or combinations of two or more domains from specific IgG isotypes for use in the methods of the invention may be based on any known parameter of the parent istoypes including affinity to FcyR (Table 7; Flesch et al. (2000) "Functions Of The Fc Receptors For Immunoglobulin G, " J. Clin. Lab. Anal. 14: 141-156; Chappel et al. (1993) "Identification Of A Secondary Fc Gamma RI Binding Site Within A Genetically Engineered Human IgG Antibody, " J. Biol. Chem.
  • Fc regions or domains from IgG isotypes known to preferentially bind Clq or FcyRIIIA e.g., IgG3 (Briiggemann et al. (1987) "Comparison Of The Effector Functions Of Human Immunoglobulins Using A Matched Set Of Chimeric Antibodies, " J. Exp. Med. 166: 1351- 1361 ), may be combined with Fc amino acid modifications of known in the art to enhance ADCC, to engineer a diabody molecule such that effector function activity, e.g., complement activation or ADCC, is maximized.
  • effector function activity e.g., complement activation or ADCC
  • the diabody molecules of the invention may be recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to heterologous polypeptides (i.e. , an unrelated polypeptide; or portion thereof, preferably at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids of the polypeptide to generate fusion proteins.
  • heterologous polypeptides i.e. , an unrelated polypeptide; or portion thereof, preferably at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids of the polypeptide to generate fusion proteins.
  • the fusion does not necessarily need to be direct, but may occur through linker sequences.
  • the diabody molecules of the invention may be conjugated to a therapeutic agent or a drug moiety that modifies a given biological response.
  • a therapeutic agent e.g. , a drug moiety that modifies a given biological response.
  • at least one binding region of the diabody may be designed to bind the therapeutic agent or desired drug moiety without affecting diabody binding.
  • Therapeutic agents or drug moieties are not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin (i.e.
  • a protein such as tumor necrosis factor, interferons including, but not limited to, a-interferon (IFN-a), ⁇ -interferon (IFN- ⁇ ), nerve growth factor (NGF), platelet derived growth factor (PDGF), tissue plasminogen activator (TP A), an apoptotic agent (e.g. , TNF- , TNF- ⁇ , AIM I as disclosed in PCT Publication No. WO 97/33899), AIM II (see, PCT Publication No. WO 97/3491 1), Fas ligand, and VEGI (PCT Publication No.
  • IFN-a a-interferon
  • IFN- ⁇ nerve growth factor
  • PDGF platelet derived growth factor
  • TP A tissue plasminogen activator
  • an apoptotic agent e.g. , TNF- , TNF- ⁇ , AIM I as disclosed in PCT Publication No. WO 97/33899
  • AIM II see, PCT
  • a thrombotic agent or an anti- angiogenic agent e.g. , angiostatin or endostatin
  • a biological response modifier such as, for example, a lymphokine (e.g. , interleukin-1 ("IL- 1 "), interleukin-2 ("IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), and granulocyte colony stimulating factor (“G-CSF”), macrophage colony stimulating factor, (“M-CSF”), or a growth factor (e.g. , growth hormone ("GH”); proteases, or ribonucleases.
  • IL-1 interleukin-1
  • IL-2 interleukin-2
  • IL-6 interleukin-6
  • GM-CSF granulocyte macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • M-CSF macrophage colony stimulating factor
  • GH growth hormone
  • the diabody molecules of the invention can be fused to marker sequences, such as a peptide to facilitate purification.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 9131 1 ), among others, many of which are commercially available.
  • a pQE vector QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 9131 1
  • hexa-histidine provides for convenient purification of the fusion protein.
  • Other peptide tags useful for purification include, but are not limited to, the hemagglutinin "HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al. (1984) "The Structure Of An Antigenic Determinant In A Protein, " Cell, 37:767-778) and the "flag” tag (Knappik et al. (1994) "An Improved Affinity Tag Based On The FLAG Peptide For The Detection And Purification Of
  • DNA shuffling may be employed to alter the activities of molecules of the invention (e.g., epitope binding sites with higher affinities and lower dissociation rates). See, generally, U.S. Patent Nos. 5,605,793; 5,81 1 ,238; 5,830,721 ; 5,834,252; and 5,837,458, and Patten et al. (1997) "Applications Of DNA Shuffling To Pharmaceuticals And Vaccines, " Curr. Opinion Biotechnol.
  • the diabody molecules of the invention, or the nucleic acids encoding the molecules of the invention may be further altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination.
  • One or more portions of a polynucleotide encoding a molecule of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
  • the present invention also encompasses diabody molecules of the invention conjugated to or immunospecifically recognizing a diagnostic or therapeutic agent or any other molecule for which serum half-life is desired to be increased/decreased and/or targeted to a particular subset of cells.
  • the molecules of the invention can be used diagnostically to, for example, monitor the development or progression of a disease, disorder or infection as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the molecules of the invention to a detectable substance or by the molecules immunospecifically
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals, and nonradioactive
  • the detectable substance may be coupled or conjugated either directly to the molecules of the invention or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art, or the molecule may immunospecifically recognize the detectable substance: immunospecifically binding said substance. See, for example, U.S. Patent No. 4,741 ,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Such diagnosis and detection can be accomplished designing the molecules to
  • detectable substances including, but not limited to, various enzymes, enzymes including, but not limited to, horseradish peroxidase, alkaline phosphatase, beta- galactosidase, or acetylcholinesterase; prosthetic group complexes such as, but not limited to, streptavidin/biotin and avidin/biotin; fluorescent materials such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent material such as, but not limited to, luminol; bioluminescent materials such as, but not limited to, luciferase, luciferin, and aequorin; radioactive material such as, but not limited to, bismuth ( Bi), carbon ( 14 C), chromium ( 51
  • the diabody molecules of the invention may immunospecifically recognize or be conjugated to a therapeutic moiety such as a cytotoxin (e.g., a cytostatic or cytocidal agent), a therapeutic agent or a radioactive element (e.g., alpha-emitters, gamma-emitters, etc.). Cytotoxins or cytotoxic agents include any agent that is detrimental to cells.
  • a cytotoxin e.g., a cytostatic or cytocidal agent
  • a therapeutic agent e.g., alpha-emitters, gamma-emitters, etc.
  • Cytotoxins or cytotoxic agents include any agent that is detrimental to cells.
  • Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 - dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambuci
  • daunorubicin (formerly daunomycin) and doxorubicin
  • antibiotics e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)
  • anti-mitotic agents e.g., vincristine and vinblastine
  • a diabody molecule of the invention can be conjugated to or be designed to immunospecifically recognize therapeutic moieties such as a radioactive materials or macrocyclic chelators useful for conjugating radiometal ions (see above for examples of radioactive materials).
  • the macrocyclic chelator is l ,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) which can be attached to the polypeptide via a linker molecule.
  • the macrocyclic chelator is l ,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) which can be attached to the polypeptide via
  • the diabody molecule of the invention may be administered with or without a therapeutic moiety conjugated to it, administered alone, or in combination with cytotoxic factor(s) and/or cytokine(s) for use as a therapeutic treatment.
  • At least one epitope of a multivalent, e.g., tetravalent, diabody molecule may be designed to immunospecifically recognize a therapeutic agent, e.g. , cytotoxic factor(s) and/or cytokine(s), which may be administered concurrently or subsequent to the molecule of the invention.
  • a therapeutic agent e.g. , cytotoxic factor(s) and/or cytokine(s)
  • the diabody molecule may specifically target the therapeutic agent in a manner similar to direct conjugation.
  • a molecule of the invention can be conjugated to an antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.
  • Diabody molecules of the invention may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen.
  • solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • the diabody molecules of the present invention may be characterized in a variety of ways.
  • molecules of the invention may be assayed for the ability to immunospecifically bind to an antigen, e.g. , FcRIIIA or FcRIIB, or, where the molecule comprises an Fc domain (or portion thereot) for the ability to exhibit Fc-FcyR interactions, i.e. specific binding of an Fc domain (or portion thereof) to an FcyR.
  • an assay may be performed in solution (e.g., Houghten (1992) "The Use Of Synthetic Peptide
  • Molecules of the invention that have been engineered to comprise multiple epitope binding domains may be assayed for immunospecific binding to one or more antigens (e.g., cancer antigen and cross-reactivity with other antigens (e.g. , FcyR)) or, where the molecules comprise am Fc domain (or portion thereof) for Fc-FcyR interactions by any method known in the art.
  • Antigens e.g., cancer antigen and cross-reactivity with other antigens (e.g. , FcyR)
  • immunospecific binding, cross-reactivity, and Fc-FcyR interactions include, but are not limited to, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), '"sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few.
  • Such assays are routine and well known in the art (see, e.g.
  • the binding affinity and the off-rate of antigen-binding domain interaction or Fc-FcyR interaction can be determined by competitive binding assays.
  • a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen, such as tetrameric FcyR (e.g., 3 H or 125 I, see Section 5.4.1) with a molecule of interest (e.g.
  • molecules of the present invention comprising multiple epitope binding domains in the presence of increasing amounts of unlabeled epitope, such as tetrameric FcyR (see Section 5.4.1), and the detection of the molecule bound to the labeled antigen.
  • unlabeled epitope such as tetrameric FcyR (see Section 5.4.1)
  • the affinity of the molecule of the present invention for an antigen and the binding off- rates can be determined from the saturation data by Scatchard analysis.
  • the affinities and binding properties of the molecules of the invention for an antigen or FcyR may be initially determined using in vitro assays (biochemical or immunological based assays) known in the art for antigen-binding domain or Fc-FcyR, interactions, including but not limited to ELISA assay, surface plasmon resonance assay, immunoprecipitation assays.
  • the binding properties of the molecules of the invention are also characterized by in vitro functional assays for determining one or more FcyR mediator effector cell functions, as described in section 5.4.2.
  • the molecules of the invention have similar binding properties in in vivo models (such as those described and disclosed herein) as those in in vitro based assays.
  • the present invention does not exclude molecules of the invention that do not exhibit the desired phenotype in in vitro based assays but do exhibit the desired phenotype in vivo.
  • screening and identifying molecules comprising multiple epitope binding domains and, optionally, Fc domains (or portions thereof) are done functional based assays, preferably in a high throughput manner.
  • the functional based assays can be any assay known in the art for characterizing one or more FcyR mediated effector cell functions such as those described herein in Sections 5.4.2 and 5.4.3.
  • Non-limiting examples of effector cell functions include but are not limited to, antibody-dependent cell mediated cytotoxicity (ADCC), antibody-dependent phagocytosis, phagocytosis, opsonization, opsonophagocytosis, cell binding, rosetting, C lq binding, and complement dependent cell mediated cytotoxicity.
  • ADCC antibody-dependent cell mediated cytotoxicity
  • phagocytosis phagocytosis
  • opsonization opsonophagocytosis
  • cell binding rosetting
  • C lq binding complement dependent cell mediated cytotoxicity
  • BIAcore kinetic analysis is used to determine the binding on and off rates of molecules of the present invention to an antigen or and FcyR.
  • BIAcore kinetic analysis comprises analyzing the binding and dissociation of an antigen or FcyR from chips with immobilized molecules (e.g., molecules comprising epitope binding domains or Fc domains (or portions thereof), respectively) on their surface.
  • immobilized molecules e.g., molecules comprising epitope binding domains or Fc domains (or portions thereof), respectively
  • FACS fluorescence activated cell sorting
  • Flow sorters are capable of rapidly examining a large number of individual cells that have been bound, e.g., opsonized, by molecules of the invention (e.g. , 10-100 million cells per hour) (Shapiro et al. (1995) Practical Flow Cytometry).
  • specific parameters used for optimization of diabody behavior include but are not limited to, antigen concentration (i.e.
  • Flow cytometers for sorting and examining biological cells are well known in the art. Known flow cytometers are described, for example, in U.S. Patent Nos. 4,347,935; 5,464,581 ; 5,483,469; 5,602,039; 5,643,796; and 6,21 1 ,477; the entire contents of which are incorporated by reference herein. Other known flow cytometers are the FACS VantageTM system manufactured by Becton Dickinson and Company, and the COPASTM system manufactured by Union Biometrica.
  • Characterization of target antigen binding affinity or Fc-FcyR binding affinity, and assessment of target antigen or FcyR density on a cell surface may be made by methods well known in the art such as Scatchard analysis or by the use of kits as per manufacturer's instructions, such as QuantumTM Simply Cellular ® (Bangs Laboratories, Inc., Fishers, IN).
  • the one or more functional assays can be any assay known in the art for characterizing one or more FcyR mediated effector cell function as known to one skilled in the art or described herein.
  • the molecules of the invention comprising multiple epitope binding domains and, optionally, and Fc domain (or portion thereof) are assayed in an ELISA assay for binding to one or more target antigens or one or more FcyRs, e.g., FcyRIIIA, FcyRIIA, FcyRIIA; followed by one or more ADCC assays.
  • the molecules of the invention are assayed further using a surface plasmon resonance-based assay, e.g., BIAcore.
  • Surface plasmon resonance-based assay e.g., BIAcore.
  • the molecules of the invetion comprising multiple epitope binding domains and, optionally, and Fc domain (or portion thereof) is further characterized in an animal model for interaction with a target antigen (e.g., an FcyR) or for Fc-FcyR interaction.
  • a target antigen e.g., an FcyR
  • Fc-FcyR interactions are to be assessed, preferred animal models for use in the methods of the invention are, for example, transgenic mice expressing human FcyRs, e.g. , any mouse model described in U.S. Patent No. 5,877,397, and 6,676,927 which are incorporated herein by reference in their entirety.
  • transgenic mice for use in such methods include, but are not limited to, nude knockout FcyRIIIA mice carrying human FcyRIIIA; nude knockout FcyRIIIA mice carrying human FcyRIIA; nude knockout FcyRIIIA mice carrying human FcyRIIB and human FcyRIIIA; nude knockout FcyRIIIA mice carrying human FcyRIIB and human FcyRIIA; nude knockout FcyRIIIA and FcyRIIA mice carrying human FcyRIIIA and FcyRIIA and nude knockout FcyRIIIA, FcyRIIA and FcyRIIB mice carrying human FcyRIIIA, FcyRIIA and FcyRIIB mice carrying human FcyRIIIA, FcyRIIA and FcyRIIB.
  • FcyRIIIA 158 V displays a higher affinity for IgGl than 158F and an increased ADCC activity (see, e.g. , Koene et al.
  • humanized anti-CD20 monoclonal antibody Rituximab was therapeutically more effective in FcyRIIIA 158V homozygous patients compared to FcyRIIIA 158F homozygous patients (See, e.g., Cartron et al. (2002) "Therapeutic Activity Of Humanized Anti-CD20
  • therapeutic molecules comprising this region may also be more effective on patients heterozygous for FcyRIIIA- 158V and FcyRIIIA- 158F, and in patients with FcyRIIA-131H.
  • selection of molecules of the invention with alternate allotypes may provide for variants that once engineered into therapeutic diabodies will be clinically more efficacious for patients homozygous for said allotype.
  • An FcyR binding assay was developed for determining the binding of the molecules of the invention to FcyR, and, in particular, for determining binding of Fc domains to FcyR.
  • the assay allowed detection and quantitation of Fc-FcyR interactions, despite the inherently weak affinity of the receptor for its ligand, e.g. , in the micromolar range for FcyRIIB and FcyRIIIA.
  • the method is described in detail in International Application WO04/063351 and U.S. Patent Application Publications 2005/0037000 and 2005/0064514, each of which is hereby incorporated by reference in its entirety. Briefly, the method involves the formation of an FcyR complex that may be sued in any standard immunoassay known in the art, e.g. , FACS, ELISA, surface plasmon resonance, etc.
  • the FcyR complex has an improved avidity for an Fc region, relative to an uncomplexed FcyR.
  • the preferred molecular complex is a tetrameric immune complex, comprising: (a) the soluble region of FcyR (e.g.
  • the soluble region of FcyRIIIA, FcyRIIA or FcyRIIB (b) a biotinylated 15 amino acid AVITAG sequence (AVITAG) operably linked to the C-terminus of the soluble region of FcyR (e.g., the soluble region of FcyRIIIA, FcyRIIA or FcyRIIB); and (c) streptavidin-phycoerythrin (SA-PE); in a molar ratio to form a tetrameric FcyR complex (preferably in a 5: 1 molar ratio).
  • AVITAG biotinylated 15 amino acid AVITAG sequence
  • the fusion protein is biotinylated enzymatically, using for example, the E.coli Bir A enzyme, a biotin ligase which specifically biotinylates a lysine residue in the 15 amino acid AVITAG sequence.
  • the biotinylated soluble FcyR proteins are then mixed with SA- PE in a IX SA-PE:5X biotinylated soluble FcyR molar ratio to form a tetrameric FcyR complex.
  • Polypeptides comprising Fc regions have been shown to bind the tetrameric FcyR complexes with at least an 8-fold higher affinity than the monomeric uncomplexed FcyR.
  • the binding of polypeptides comprising Fc regions to the tetrameric FcyR complexes may be determined using standard techniques known to those skilled in the art, such as for example, fluorescence activated cell sorting (FACS), radioimmunoassays, ELISA assays, etc.
  • the invention encompasses the use of the immune complexes comprising molecules of the invention, and formed according to the methods described above, for determining the functionality of molecules comprising an Fc region in cell-based or cell-free assays.
  • the reagents may be provided in an assay kit, i.e., a packaged combination of reagents for assaying the ability of molecules comprising Fc regions to bind FcyR tetrameric complexes.
  • an assay kit i.e., a packaged combination of reagents for assaying the ability of molecules comprising Fc regions to bind FcyR tetrameric complexes.
  • Other forms of molecular complexes for use in determining Fc-FcyR interactions are also contemplated for use in the methods of the invention, e.g. , fusion proteins formed as described in U.S. Provisional Application 60/439,709, filed on January 13, 2003; which is incorporated herein by reference in its entirety.
  • the invention encompasses characterization of the molecules of the invention comprising multiple epitope binding domains and, optionally, Fc domains (or portions thereof) using assays known to those skilled in the art for identifying the effector cell function of the molecules.
  • the invention encompasses characterizing the molecules of the invention for FcyR-mediated effector cell function.
  • binding of the FcyR by the diabody molecule may serve to activate FcyR-mediated pathways similar to those activated by FcyR-Fc binding.
  • the diabody molecule may elicit FcyR-mediated effector cell function without containing an Fc domain (or portion thereof), or without concomitant Fc-FcyR binding.
  • effector cell functions include but are not limited to, antibody-dependent cell mediated cytotoxicity, phagocytosis, opsonization,
  • opsonophagocytosis opsonophagocytosis, Clq binding, and complement dependent cell mediated cytotoxicity.
  • Any cell-based or cell free assay known to those skilled in the art for determining effector cell function activity can be used (For effector cell assays, see Perussia et al. (2000) "Assays For Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) And Reverse ADCC (Redirected Cytotoxicity) In Human Natural Killer Cells, " Methods Mol. Biol. 121 : 179-92; Baggiolini et al.
  • the molecules of the invention can be assayed for
  • the FcyR-mediated phagocytosis of the molecules of the invention may be assayed in other phagocytes, e.g. , neutrophils (polymorphonuclear leuckocytes; PMN); human peripheral blood monocytes, monocyte-derived macrophages, which can be obtained using standard procedures known to those skilled in the art (e.g. , see Brown (1994) "In Vitro Assays Of Phagocytic Function Of Human Peripheral Blood Leukocytes: Receptor Modulation And Signal Transduction, " Methods Cell Biol., 45: 147-164).
  • neutrophils polymorphonuclear leuckocytes
  • monocyte-derived macrophages which can be obtained using standard procedures known to those skilled in the art (e.g. , see Brown (1994) "In Vitro Assays Of Phagocytic Function Of Human Peripheral Blood Leukocytes: Receptor Modulation And Signal Transduction, " Methods Cell Biol., 45: 147
  • the function of the molecules of the invention is characterized by measuring the ability of THP-1 cells to phagocytose fluoresceinated IgG-opsonized sheep red blood cells (SRBC) by methods previously described (Tridandapani et al. (2000) "The Adapter Protein L AT Enhances Fcgamma Receptor-Mediated Signal Transduction In Myeloid Cells, " J. Biol. Chem. 275: 20480- 20487).
  • SRBC phagocytose fluoresceinated IgG-opsonized sheep red blood cells
  • ADCP antibody-dependent opsonophagocytosis assay
  • a target bioparticle such as Escherichia coli- labeled FITC (Molecular Probes) or Staphylococcus aureus-FYTC with (i) wild-type 4-4- 20 antibody, an antibody to fluorescein (See Bedzyk et al. (1989) "Comparison Of Variable Region Primary Structures Within An Anti-Fluorescein Idiotype Family, " J. Biol.
  • THP-1 cells used in the assay are analyzed by FACS for expression of FcyR on the cell surface.
  • THP-1 cells express both CD32A and CD64.
  • CD64 is a high affinity FcyR that is blocked in conducting the ADCP assay in accordance with the methods of the invention.
  • the THP-1 cells are preferably blocked with 100 ⁇ tg/mL soluble IgGl or 10% human serum.
  • the gate is preferably set on THP-1 cells and median fluorescence intensity is measured.
  • the ADCP activity for individual mutants is calculated and reported as a normalized value to the wild type chMab 4-4-20 obtained.
  • the opsonized particles are added to THP-1 cells such that the ratio of the opsonized particles to THP-1 cells is 30: 1 or 60: 1 .
  • the ADCP assay is conducted with controls, such as E. coli-YYYC in medium, E.
  • the molecules of the invention can be assayed for FcyR-mediated ADCC activity in effector cells, e.g., natural killer cells, using any of the standard methods known to those skilled in the art (See e.g., Perussia et al. (2000) "Assays For Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) And Reverse
  • An exemplary assay for determining ADCC activity of the molecules of the invention is based on a 51 Cr release assay comprising of: labeling target cells with [ 51 Cr]Na 2 Cr0 4 (this cell-membrane permeable molecule is commonly used for labeling since it binds cytoplasmic proteins and although spontaneously released from the cells with slow kinetics, it is released massively following target cell necrosis); opsonizing the target cells with the molecules of the invention comprising variant heavy chains; combining the opsonized radiolabeled target cells with effector cells in a microtitre plate at an appropriate ratio of target cells to effector cells; incubating the mixture of cells for 16-18 hours at 37°C; collecting supernatants; and analyzing radioactivity.
  • % lysis (experimental cpm - target leak cpm)/(detergent lysis cpm - target leak cpm) x 100%.
  • % lysis (ADCC-AICC)/(maximum release-spontaneous release).
  • a graph can be generated by varying either the target: effector cell ratio or antibody concentration.
  • the effector cells used in the ADCC assays of the invention are peripheral blood mononuclear cells (PBMC) that are preferably purified from normal human blood, using standard methods known to one skilled in the art, e.g. , using Ficoll- Paque density gradient centrifugation.
  • PBMC peripheral blood mononuclear cells
  • Preferred effector cells for use in the methods of the invention express different FcyR activating receptors.
  • the invention encompasses, effector cells, THP-1 , expressing FcyRI, FcyRIIA and FcyRIIB, and monocyte derived primary macrophages derived from whole human blood expressing both FcyRIIIA and FcyRIIB, to determine if heavy chain antibody mutants show increased ADCC activity and phagocytosis relative to wild type IgGl antibodies.
  • the human monocyte cell line, THP-1 activates phagocytosis through expression of the high affinity receptor FcyRI and the low affinity receptor FcyRIIA (Fleit et al. (1991) "The Human Monocyte-Like Cell Line THP-1 Expresses Fc Gamma RI And Fc Gamma RII, " J. Leuk. Biol. 49: 556-565). THP-1 cells do not constitutively express FcyRIIA or FcyRIIB. Stimulation of these cells with cytokines affects the FcR expression pattern (Pricop et al.
  • the presence or absence of receptors on the cell surface can be determined by FACS using common methods known to one skilled in the art. Cytokine induced expression of FcyR on the cell surface provides a system to test both activation and inhibition in the presence of FcyRIIB. If THP-1 cells are unable to express the FcyRIIB the invention also encompasses another human monocyte cell line, U937. These cells have been shown to terminally differentiate into macrophages in the presence of IFNy and TNF (Koren et al. (1979) "In Vitro Activation Of A Human Macrophage -Like Cell Line, " Nature 279: 328-331 ).
  • FcyR dependent tumor cell killing is mediated by macrophage and NK cells in mouse tumor models (Clynes et al. (1998) "Fc Receptors Are Required In Passive And Active Immunity To Melanoma, " Proc. Nat. Acad. Sci. USA 95: 652-656).
  • the invention encompasses the use of elutriated monocytes from donors as effector cells to analyze the efficiency Fc mutants to trigger cell cytotoxicity of target cells in both phagocytosis and ADCC assays. Expression patterns of FcyRI, FcyRIIIA, and FcyRIIB are affected by different growth conditions.
  • FcyR expression from frozen elutriated monocytes, fresh elutriated monocytes, monocytes maintained in 10% FBS, and monocytes cultured in FBS + GM-CSF and or in human serum may be determined using common methods known to those skilled in the art. For example, cells can be stained with FcyR specific antibodies and analyzed by FACS to determine FcR profiles. Conditions that best mimic
  • macrophage in vivo FcyR expression is then used for the methods of the invention.
  • the invention encompasses the use of mouse cells especially when human cells with the right FcyR profiles are unable to be obtained. In some embodiments, the invention encompasses the mouse macrophage cell line
  • transfectants isolated using methods known in the art, see, e.g., Ralph et al. (1977) "Antibody-Dependent Killing Of Erythrocyte And Tumor Targets By Macrophage-Related Cell Lines: Enhancement By PPD And LPS, " J. Immunol. 1 19: 950-4). Transfectants can be quantitated for FcyRIIIA expression by FACS analysis using routine experimentation and high expressors can be used in the ADCC assays of the invention. In other embodiments, the invention encompasses isolation of spleen peritoneal macrophage expressing human FcyR from knockout transgenic mice such as those disclosed herein.
  • Lymphocytes may be harvested from peripheral blood of donors (PBM) using a Ficoll-Paque gradient (Pharmacia). Within the isolated mononuclear population of cells the majority of the ADCC activity occurs via the natural killer cells (NK) containing FcyRIIIA but not FcyRIIB on their surface. Results with these cells indicate the efficacy of the mutants on triggering NK cell ADCC and establish the reagents to test with elutriated monocytes.
  • PBM peripheral blood of donors
  • NK natural killer cells
  • Target cells used in the ADCC assays of the invention include, but are not limited to, breast cancer cell lines, e.g., SK-BR-3 with ATCC accession number HTB-30 (see, e.g. , Tremp et al. ( 1976) "Human Breast Cancer In Culture, " Recent Results Cancer Res. 33-41 ); B-lymphocytes; cells derived from Burkitts lymphoma, e.g., Raji cells with ATCC accession number CCL-86 (see, e.g., Epstein et al. (1965) "Characteristics And Mode Of Growth Of Tissue Culture Strain (EBl) Of Human Lymphoblasts From Burkitt's Lymphoma. " J. Natl.
  • the ADCC assay is based on the ability of N cells to mediate cell death via an apoptotic pathway. NK cells mediate cell death in part by FcyRIIIA's recognition of an IgG Fc domain bound to an antigen on a cell surface.
  • the ADCC assays used in accordance with the methods of the invention may be radioactive based assays or fluorescence based assays.
  • the ADCC assay used to characterize the molecules of the invention comprising variant Fc regions comprises labeling target cells, e.g., SK-BR-3, MCF-7, OVCAR3, Raji, Daudi cells, opsonizing target cells with an antibody that recognizes a cell surface receptor on the target cell via its antigen binding site; combining the labeled opsonized target cells and the effector cells at an appropriate ratio, which can be determined by routine experimentation; harvesting the cells; detecting the label in the supernatant of the lysed target cells, using an appropriate detection scheme based on the label used.
  • the target cells may be labeled either with a radioactive label or a fluorescent label, using standard methods known in the art.
  • the labels include, but are not limited to, [ Cr]Na 2 Cr0 4 ; and the acetoxymethyl ester of the fluorescence enhancing ligand, 2,2 , :6 ⁇ 2"-te yridine-6-6"-dicarbo ylate (TDA).
  • a time resolved fluorimetric assay is used for measuring ADCC activity against target cells that have been labeled with the acetoxymethyl ester of the fluorescence enhancing ligand, 2,2' :6',2"-terpyridine-6- 6"-dicarboxylate (TDA).
  • fluorimetric assays are known in the art, e.g. , see, Blomberg et al. (1996) "Time-Resolved Fluorometric Assay For Natural Killer Activity Using Target Cells Labelled With A Fluorescence Enhancing Ligand, " Journal of Immunological Methods, 193: 199-206; which is incorporated herein by reference in its entirety.
  • target cells are labeled with the membrane permeable acetoxymethyl diester of TDA (bis(acetoxymethyl) 2,2':6 ⁇ 2"-terpyridine-6-6"-dicarboxylate, (BATDA), which rapidly diffuses across the cell membrane of viable cells.
  • TDA bis(acetoxymethyl) 2,2':6 ⁇ 2"-terpyridine-6-6"-dicarboxylate, (BATDA)
  • BATDA bis(acetoxymethyl) 2,2':6 ⁇ 2"-terpyridine-6-6"-dicarboxylate
  • the TDA released from the lysed target cells is chelated with Eu3+ and the fluorescence of the Europium-TDA chelates formed is quantitated in a time-resolved fluorometer (e.g. , Victor 1420, Perkin Elmer/Wallace).
  • a time-resolved fluorometer e.g. , Victor 1420, Perkin Elmer/Wallace.
  • the ADCC assay used to characterize the molecules of the invention comprising multiple epitope binding sites and, optionally, an Fc domain (or portion thereof) comprises the following steps: Preferably 4-5 10 6 target cells (e.g., SK-BR-3, MCF-7, OVCAR3, Raji cells) are labeled with bis(acetoxymethyl) 2,2' :6',2"-terpyridine-t-6"-dicarboxylate (DELFIA BATDA Reagent, Perkin
  • the number of target cells used in the ADCC assay should preferably not exceed 5xl0 6 .
  • BATDA reagent is added to the cells and the mixture is incubated at 37°C preferably under 5% C0 2 , for at least 30 minutes.
  • the cells are then washed with a physiological buffer, e.g. , PBS with 0.125 mM sulfinpyrazole, and media containing 0.125 mM sulfinpyrazole.
  • the labeled target cells are then opsonized (coated) with a molecule of the invention comprising an epitope binding domain specific for FcyRIIA and, optionally, an Fc domain (or portion thereof).
  • the molecule used in the ADCC assay is also specific for a cell surface receptor, a tumor antigen, or a cancer antigen.
  • the diabody molecule of the invetion may specifically bind any cancer or tumor antigen, such as those listed in section 5.6.1.
  • the target cells in the ADCC assay are chosen according to the epitope binding sites engineered into the diabody of the invention, such that the diabody binds a cell surface receptor of the target cell specifically.
  • Target cells are added to effector cells, e.g., PBMC, to produce
  • effectontarget ratios of approximately 1 : 1 , 10: 1 , 30: 1 , 50: 1 , 75: 1 , or 100: 1 .
  • the effector and target cells are incubated for at least two hours, up to 3.5 hours, at 37°C, under 5% C0 2 .
  • Cell supernatants are harvested and added to an acidic europium solution (e.g. , DELFIA Europium Solution, Perkin Elmer/Wallac).
  • an acidic europium solution e.g. , DELFIA Europium Solution, Perkin Elmer/Wallac.
  • Europium-TDA chelates formed is quantitated in a time-resolved fluorometer (e.g. , Victor 1420, Perkin Elmer/Wallac). Maximal release (MR) and spontaneous release (SR) are determined by incubation of target cells with 1 % TX-100 and media alone, respectively. Antibody independent cellular cytotoxicity (AICC) is measured by incubation of target and effector cells in the absence of a test molecule, e.g., diabody of the invention. Each assay is preferably performed in triplicate. The mean percentage specific lysis is calculated as: Experimental release (ADCC) - AICC)/(MR-SR) x 100.
  • ADCC Experimental release
  • AICC AICC
  • the invention encompasses assays known in the art, and exemplified herein, to characterize the binding of Clq and mediation of complement dependent cytotoxicity (CDC) by molecules of the invention comprising Fc domains (or portions thereof).
  • a Clq binding ELISA may be performed.
  • An exemplary assay may comprise the following: assay plates may be coated overnight at 4C with polypeptide comprising a molecule of the invention or starting polypeptide (control) in coating buffer. The plates may then be washed and blocked. Following washing, an aliquot of human Clq may be added to each well and incubated for 2 hrs at room temperature.
  • a sheep anti-complement Clq peroxidase conjugated antibody may be added to each well and incubated for 1 hour at room temperature.
  • the plate may again be washed with wash buffer and 100 ul of substrate buffer containing OPD (O-phenylenediamine dihydrochloride (Sigma)) may be added to each well.
  • OPD O-phenylenediamine dihydrochloride (Sigma)
  • the oxidation reaction observed by the appearance of a yellow color, may be allowed to proceed for 30 minutes and stopped by the addition of 100 ul of 4.5 NH2 S04.
  • the absorbance may then read at (492-405) nm.
  • a complement dependent cytotoxicity (CDC) assay may be performed, e.g. as described in Gazzano-Santoro et al. (1997) "A Non-Radioactive Complement-Dependent Cytotoxicity Assay For Anti-CD20 Monoclonal Antibody, " J. Immunol. Methods 202: 163-171 , which is incorporated herein by reference in its entirety. Briefly, various concentrations of the molecule comprising a (variant) Fc domain (or portion thereof) and human complement may be diluted with buffer. Cells which express the antigen to which the diabody molecule binds may be diluted to a density of about lxlO 6 cells/ml.
  • Mixtures of the diabody molecules comprising a (variant) Fc domain (or portion thereof), diluted human complement and cells expressing the antigen may be added to a flat bottom tissue culture 96 well plate and allowed to incubate for 2 hrs at 37°C. and 5% C02 to facilitate complement mediated cell lysis. 50 uL of alamar blue (Accumed International) may then be added to each well and incubated overnight at 37°C. The absorbance is measured using a 96-well fluorometer with excitation at 530 nm and emission at 590 nm. The results may be expressed in relative fluorescence units (RFU).
  • REU relative fluorescence units
  • sample concentrations may be computed from a standard curve and the percent activity as compared to nonvariant molecule, i.e., a molecule not comprising an Fc domain or comprising a non-variant Fc domain, is reported for the variant of interest.
  • the molecules of the invention comprising multiple epitope binding domain and, optionally, an Fc domain may be assayed using any surface plasmon resonance based assays known in the art for characterizing the kinetic parameters of an antigen-binding domain or Fc-FcyR binding.
  • SPR instrument commercially available including, but not limited to, BIAcore Instruments, available from Biacore AB (Uppsala, Sweden); IAsys instruments available from Affinity Sensors (Franklin, MA.); IBIS system available from Windsor Scientific Limited (Berks, UK), SPR-CELLIA systems available from Nippon Laser and Electronics Lab (Hokkaido, Japan), and SPR Detector Spreeta available from Texas Instruments (Dallas, TX) can be used in the instant invention.
  • BIAcore Instruments available from Biacore AB (Uppsala, Sweden
  • IAsys instruments available from Affinity Sensors (Franklin, MA.)
  • IBIS system available from Windsor Scientific Limited (Berks, UK)
  • SPR-CELLIA systems available from Nippon Laser and Electronics Lab (Hokkaido, Japan)
  • SPR Detector Spreeta available from Texas Instruments (Dallas, TX)
  • SPR based assays involve immobilizing a member of a binding pair on a surface, and monitoring its interaction with the other member of the binding pair in solution in real time.
  • SPR is based on measuring the change in refractive index of the solvent near the surface that occurs upon complex formation or dissociation.
  • the surface onto which the immobilization occurs is the sensor chip, which is at the heart of the SPR technology; it consists of a glass surface coated with a thin layer of gold and forms the basis for a range of specialized surfaces designed to optimize the binding of a molecule to the surface.
  • a variety of sensor chips are commercially available especially from the companies listed supra, all of which may be used in the methods of the invention.
  • sensor chips examples include those available from BIAcore AB, Inc., e.g., Sensor Chip CM5, SA, NTA, and HPA.
  • a molecule of the invention may be immobilized onto the surface of a sensor chip using any of the immobilization methods and chemistries known in the art, including but not limited to, direct covalent coupling via amine groups, direct covalent coupling via sulfhydryl groups, biotin attachment to avidin coated surface, aldehyde coupling to carbohydrate groups, and attachment through the histidine tag with NTA chips.
  • the kinetic parameters of the binding of molecules of the invention comprising multiple epitope binding sites and, optionally, and Fc domain, to an antigen or an FcyR may be determined using a BIAcore instrument (e.g. , BIAcore instrument 1000, BIAcore Inc., Piscataway, NJ).
  • a BIAcore instrument e.g. , BIAcore instrument 1000, BIAcore Inc., Piscataway, NJ.
  • any FcyR can be used to assess the binding of the molecules of the invention either where at least one epitope binding site of the diabody molecule immunospecifically recognizes an FcyR, and/or where the diabody molecule comprises an Fc domain (or portion thereof).
  • the FcyR is FcyRIIIA, preferably a soluble monomeric
  • the soluble monomeric FcyRIIIA is the extracellular region of FcyRIIIA joined to the linker- AVITAG sequence (see, U.S.
  • the FcyR is FcyRIIB, preferably a soluble dimeric FcyRIIB.
  • the soluble dimeric FcyRIIB protein is prepared in accordance with the methodology described in U.S. Provisional application No. 60/439,709 filed on January 13, 2003, which is incorporated herein by reference in its entirety.
  • FcyR recognition/binding by a molecule of the invention may be effected by multiple domains: in certain embodiments, molecules of the invention immunospecifically recognize an FcyR via one of the multiple epitope binding domains; in yet other embodiments, where the molecule of the invention comprises an Fc domain (or portion thereof), the diabody molecule may
  • the diabody molecule may recognize an FcyR via one or both of an epitope binding domain and the Fc domain (or portion thereof).
  • An exemplary assay for determining the kinetic parameters of a molecule comprising multiple epitope binding domains and, optionally, and Fc domain (or portion thereof) to an antigen and/or an FcyR using a BIAcore instrument comprises the following: a first antigen is immobilized on one of the four flow cells of a sensor chip surface, preferably through amine coupling chemistry such that about 5000 response units (RU) of said first antigen is immobilized on the surface.
  • RU response units
  • Levels of molecules of the invention bound to the surface at this stage typically ranges between 400 and 700 RU.
  • dilution series of a second antigen e.g., FcyR
  • FcyR receptor in HBS-P buffer (20mM HEPES, 150 mM NaCl, 3mM EDTA, pH 7.5) are injected onto the surface at 100 ⁇ / ⁇
  • Regeneration of molecules between different second antigen or receptor dilutions is carried out preferably by single 5 second injections of lOOmM NaHC0 3 pH 9.4; 3M NaCl. Any regeneration technique known in the art is contemplated in the method of the invention.
  • the resulting binding curves are globally fitted using computer algorithms supplied by the SPR instrument manufacturer, e.g., BIAcore, Inc. (Piscataway, NJ). These algorithms calculate both the K on and 0f f, from which the apparent equilibrium binding constant, 3 ⁇ 4 is deduced as the ratio of the two rate constants (i.e., K 0ft /K on ). More detailed treatments of how the individual rate constants are derived can be found in the BIAevaluaion Software Handbook (BIAcore, Inc., Piscataway, NJ). The analysis of the generated data may be done using any method known in the art.
  • the kinetic parameters determined using an SPR analysis may be used as a predictive measure of how a molecule of the invention will function in a functional assay, e.g. , ADCC.
  • An exemplary method for predicting the efficacy of a molecule of the invention based on kinetic parameters obtained from an SPR analysis may comprise the following: determining the 0ff values for binding of a molecule of the invention to FcyRIIIA and FcyRIIB (via an epitope binding domain and/or an Fc domain (or portion thereof)); plotting (1 ) K off (wt)/Ko ff (mut) for FcyRIIIA; (2) Ko ff (mut)/K off (wt) for FcyRIIB against the ADCC data. Numbers higher than one show a decreased dissociation rate for FcyRIIIA and an increased dissociation rate for FcyRIIB relative to wild type; and possess and enhanced ADCC function.
  • the diabody molecules of the present invention can be produced using a variety of methods well known in the art, including de novo protein synthesis and recombinant expression of nucleic acids encoding the binding proteins.
  • the desired nucleic acid sequences can be produced by recombinant methods (e.g., PCR mutagenesis of an earlier prepared variant of the desired polynucleotide) or by solid-phase DNA synthesis. Usually recombinant expression methods are used.
  • the invention provides a polynucleotide that comprises a sequence encoding a CD16A VH and/or VL; in another aspect, the invention provides a polynucleotide that comprises a sequence encoding a CD32B VH and/or VL. Because of the degeneracy of the genetic code, a variety of nucleic acid sequences encode each immunoglobulin amino acid sequence, and the present invention includes all nucleic acids encoding the binding proteins described herein.
  • the present invention also includes polynucleotides that encode the molecules of the invention, including the polypeptides and antibodies.
  • polynucleotides encoding the molecules of the invention may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art.
  • nucleotide sequence of the molecules that are identified by the methods of the invention may be manipulated using methods well known in the art, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et ⁇ ,
  • human libraries or any other libraries available in the art can be screened by standard techniques known in the art, to clone the nucleic acids encoding the molecules of the invention.
  • nucleic acid sequence encoding molecules of the invention ⁇ i.e., antibodies
  • the vector for the production of the molecules may be produced by recombinant DNA technology using techniques well known in the art.
  • An expression vector comprising the nucleotide sequence of a molecule identified by the methods of the invention can be transferred to a host cell by conventional techniques ⁇ e.g. , electroporation, liposomal transfection, and calcium phosphate precipitation) and the transfected cells are then cultured by conventional techniques to produce the molecules of the invention.
  • the expression of the molecules of the invention is regulated by a constitutive, an inducible or a tissue, specific promoter.
  • the host cells used to express the molecules identified by the methods of the invention may be either bacterial cells such as Escherichia coli, or, preferably, eukaryotic cells, especially for the expression of whole recombinant immunoglobulin molecule.
  • mammalian cells such as Chinese hamster ovary cells (CHO)
  • CHO Chinese hamster ovary cells
  • a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for immunoglobulins (Foecking et a/. (1986) "Powerful And Versatile Enhancer-Promoter Unit For
  • host-expression vector systems may be utilized to express the molecules identified by the methods of the invention.
  • Such host-expression systems represent vehicles by which the coding sequences of the molecules of the invention may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express the molecules of the invention in situ.
  • These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing coding sequences for the molecules identified by the methods of the invention; yeast (e.g.
  • Saccharomyces pichia transformed with recombinant yeast expression vectors containing sequences encoding the molecules identified by the methods of the invention; insect cell systems infected with recombinant virus expression vectors (e.g., baclovirus) containing the sequences encoding the molecules identified by the methods of the invention; plant cell systems infected with recombinant virus expression vectors (e.g. , cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV) or transformed with recombinant plasmid expression vectors (e.g. , Ti plasmid) containing sequences encoding the molecules identified by the methods of the invention; or mammalian cell systems (e.g.
  • vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • vectors include, but are not limited, to the E. coli expression vector pUR278 (Riither et al. (1983) "Easy Identification OfcDNA Clones, " EMBO J. 2: 1791 - 1794), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye et al.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione- agarose beads followed by elution in the presence of free glutathione.
  • GST glutathione S-transferase
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • AcNPV is used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • the antibody coding sequence may be cloned individually into nonessential regions ⁇ e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter ⁇ e.g., the polyhedrin promoter).
  • a number of viral-based expression systems may be utilized.
  • the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination.
  • Insertion in a non-essential region of the viral genome ⁇ e.g. , region E l or E3) will result in a recombinant virus that is viable and capable of expressing the immunoglobulin molecule in infected hosts ⁇ e.g. , see Logan et al. (1984) "Adenovirus Tripartite Leader Sequence Enhances Translation Of mRNAs Late After Infection, " Proc. Natl. Acad. Sci. USA 81 :3655-3659).
  • Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences.
  • initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert.
  • exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic.
  • the efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bitter et al. (1987) "Expression And Secretion Vectors For Yeast, " Methods in Enzymol. 153:516- 544).
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
  • the polypeptides comprising a diabody molecule of the invention may be expressed as a single gene product (e.g. , as a single polypeptide chain, i.e., as a polyprotein precursor), requiring proteolytic cleavage by native or recombinant cellular mechanisms to form the separate polypeptides of the diabody molecules of the invention.
  • the invention thus encompasses engineering a nucleic acid sequence to encode a polyprotein precursor molecule comprising the polypeptides of the invention, which includes coding sequences capable of directing post translational cleavage of said polyprotein precursor.
  • Post-translational cleavage of the polyprotein precursor results in the polypeptides of the invention.
  • the post translational cleavage of the precursor molecule comprising the polypeptides of the invention may occur in vivo (i.e., within the host cell by native or recombinant cell systems/mechanisms, e.g. furin cleavage at an appropriate site) or may occur in vitro (e.g. incubation of said polypeptide chain in a composition comprising proteases or peptidases of known activity and/or in a composition comprising conditions or reagents known to foster the desired proteolytic action).
  • proteases or peptidases known in the art can be used for the described modification of the precursor molecule, e.g., thrombin (which recognizes the amino acid sequence LVPR GS (SEQ ID NO: 89)), or factor Xa (which recognizes the amino acid sequence I(E/D)GR A (SEQ ID NO: 90) (Nagai et al. (1985) "Oxygen Binding Properties Of Human Mutant Hemoglobins Synthesized In Escherichia Coli, " Proc. Nat. Acad. Sci.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • mammalian host cells include but are not limited to CHO, VERY, BHK, HeLa, COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 and Hs578Bst.
  • stable expression For long-term, high-yield production of recombinant proteins, stable expression is preferred.
  • cell lines which stably express an antibody of the invention may be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements ⁇ e.g., promoter, enhancer, sequences, transcription
  • engineered cells may be allowed to grow for 1 -2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This method may advantageously be used to engineer cell lines which express the antibodies of the invention.
  • Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the molecules of the invention.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al. (1977) "Transfer Of Purified Herpes Virus Thymidine Kinase Gene To Cultured Mouse Cells, " Cell 1 1 : 223-232), hypoxanthine-guanine phosphoribosyltransferase (Szybalska et al.
  • antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al. (1980) "Transformation Of Mammalian Cells With An Amplifiable Dominant- Acting Gene, " Proc. Natl. Acad. Sci. USA 77:3567- 3570; O'Hare et al. (1981) "Transformation Of Mouse Fibroblasts To Methotrexate Resistance By A Recombinant Plasmid Expressing A Prokaryotic Dihydrofolate
  • the expression levels of a molecule of the invention can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press. New York, 1987).
  • vector amplification for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press. New York, 1987).
  • the host cell may be co-transfected with two expression vectors of the invention, the first vector encoding the first polypeptide of the diabody molecule and the second vector encoding the second polypeptide of the diabody molecule.
  • the two vectors may contain identical selectable markers which enable equal expression of both polypeptides.
  • a single vector may be used which encodes both
  • polypeptides are polypeptides.
  • the coding sequences for the polypeptides of the molecules of the invention may comprise cDNA or genomic DNA.
  • a molecule of the invention i.e. , diabodies
  • it may be purified by any method known in the art for purification of polypeptides, polyproteins or diabodies (e.g. , analogous to antibody purification schemes based on antigen selectivity) for example, by chromatography (e.g. , ion exchange, affinity, particularly by affinity for the specific antigen (optionally after Protein A selection where the diabody molecule comprises an Fc domain (or portion thereof)), and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of polypeptides, polyproteins or diabodies.
  • chromatography e.g. , ion exchange, affinity, particularly by affinity for the specific antigen (optionally after Protein A selection where the diabody molecule comprises an Fc domain (or portion thereof)
  • centrifugation e.g. ion exchange, affinity, particularly by affinity for the specific antigen (optionally after Protein A selection where the dia
  • the molecules of the invention are particularly useful for the treatment and/or prevention of a disease, disorder or infection where an effector cell function (e.g. , ADCC) mediated by FcyR is desired (e.g., cancer, infectious disease).
  • an effector cell function e.g. , ADCC
  • FcyR mediated by FcyR
  • the diabodies of the invetion may exhibit antibody-like functionality in eliciting effector function although the diabody molecule does not comprise and Fc domain.
  • the diabody molecule may exibit FcyR binding and activity analogous to Fc-FcyR interactions.
  • molecules of the invention may bind a cell surface antigen and an FcyR (e.g.
  • the diabody molecule of the invention comprises an Fc domain (or portion thereof).
  • the Fc domain may further comprise at least one amino acid modification relative to a wild-type Fc domain (or portion thereof) and/or may comprise domains from one or more IgG isotypes (e.g., IgGl , IgG2, IgG3 or IgG4).
  • Molecules of the invetion comprising variant Fc domains may exhibit conferred or altered phenotypes relative to molecules comprising the wild type Fc domain such as an altered or conferred effector function activity (e.g. , as assayed in an NK dependent or macrophage dependent assay).
  • molecules of the invention with conferred or altered effector function activity are useful for the treatment and/or prevention of a disease, disorder or infection where an enhanced efficacy of effector function activity is desired.
  • the diabody molecules of the invention comprising an Fc domain (or portion thereof) mediate complement dependent cascade. Fc domain variants identified as altering effector function are disclosed in International Application WO04/063351 , U.S. Patent Application Publications
  • the invention encompasses methods and compositions for treatment, prevention or management of a cancer in a subject, comprising administering to the subject a therapeutically effective amount of one or more molecules comprising one or more epitope binding sites, and optionally, an Fc domain (or portion thereof) engineered in accordance with the invention, which molecule further binds a cancer antigen.
  • Molecules of the invention are particularly useful for the prevention, inhibition, reduction of growth or regression of primary tumors, metastasis of cancer cells, and infectious diseases.
  • molecules of the invention mediate effector function resulting in tumor clearance, tumor reduction or a combination thereof.
  • the diabodies of the invention mediate therapeutic activity by cross-linking of cell surface antigens and/or receptors and enhanced apoptosis or negative growth regulatory signaling.
  • the diabody molecules of the invention exhibit enhanced therapeutic efficacy relative to therapeutic antibodies known in the art, in part, due to the ability of diabody to
  • a target cell which expresses a particular antigen (e.g., FcyR) at reduced levels, for example, by virtue of the ability of the diabody to remain on the target cell longer due to an improved avidity of the diabody-epitope interaction.
  • a particular antigen e.g., FcyR
  • the diabodies of the invention with enhanced affinity and avidity for antigens are particularly useful for the treatment, prevention or management of a cancer, or another disease or disorder, in a subject, wherein the FcyRs are expressed at low levels in the target cell populations.
  • FcyR expression in cells is defined in terms of the density of such molecules per cell as measured using common methods known to those skilled in the art.
  • the molecules of the invention comprising multiple epitope binding sites and, optionally, and FcyR (or portion thereof) preferably also have a conferred or an enhanced avidity and affinity and/or effector function in cells which express a target antigen, e.g., a cancer antigen, at a density of 30,000 to 20,000 molecules/cell, at a density of 20,000 to 10,000 molecules/cell, at a density of 10,000 molecules/cell or less, at a density of 5000 molecules/cell or less, or at a density of 1000 molecules /cell or less.
  • a target antigen e.g., a cancer antigen
  • the molecules of the invention have particular utility in treatment, prevention or management of a disease or disorder, such as cancer, in a sub-population, wherein the target antigen is expressed at low levels in the target cell population.
  • the molecules of the invention may also be advantageously utilized in combination with other therapeutic agents known in the art for the treatment or prevention of diseases, such as cancer, autoimmune disease, inflammatory disorders, and infectious diseases.
  • molecules of the invention may be used in
  • the molecules of the invention may also be advantageously utilized in combination with one or more drugs used to treat a disease, disorder, or infection such as, for example anti-cancer agents, anti-inflammatory agents or anti-viral agents, e.g., as detailed in Section 5.7.
  • the invention encompasses methods and compositions for treatment or prevention of cancer in a subject comprising administering to the subject a therapeutically effective amount of one or more molecules comprising multiple epitope binding domains.
  • the invention encompasses methods and compositions for the treatment or prevention of cancer in a subject with FcyR polymorphisms such as those homozygous for the FyRIIIA-l 58V or FcyRIIIA- 158F alleles.
  • the invention encompasses engineering at least one epitope binding domain of the diabody molecule to immunospecifically bind FcyRIIIA (158F).
  • the invention encompasses engineering at least one epitope binding domain of the diabody molecule to immunospecifically bind FcyRIIIA (158V).
  • the methods of the invention encompass engineering molecules to immunospecifically recognize the low affinity activating receptors, allowing the molecules to be designed for a specific polymorphism.
  • the molecule of the invention may be engineered to comprise a variant Fc domain that exhibits enhanced affinity to FcyR (relative to a wild type Fc domain) on effector cells.
  • the engineered molecules of the invention provide better immunotherapy reagents for patients regardless of their FcyR polymorphism.
  • Diabody molecules engineered in accordance with the invention are tested by ADCC using either a cultured cell line or patient derived PMBC cells to determine the ability of the Fc mutations to enhance ADCC.
  • Standard ADCC is performed using methods disclosed herein. Lymphocytes are harvested from peripheral blood using a Ficoll-Paque gradient (Pharmacia). Target cells, i.e., cultured cell lines or patient derived cells, are loaded with Europium (PerkinElmer) and incubated with effectors for 4 hrs at 37°C. Released Europium is detected using a fluorescent plate reader (Wallac).
  • the resulting ADCC data indicates the efficacy of the molecules of the invention to trigger NK cell mediated cytotoxicity and establish which molecules can be tested with both patient samples and elutriated monocytes.
  • Diabody molecules showing the greatest potential for eliciting ADCC activity are then tested in an ADCC assay using PBMCs from patients. PBMC from healthy donors are used as effector cells.
  • the invention provides methods of preventing or treating cancer characterized by a cancer antigen by engineering the diabody molecule to immunospecifically recognize said cancer antigen such that the diabody molecule is itself cytotoxic (e.g., via crosslinking of surface receptors leading to increased apoptosis or downregulation of proliferative signals) and/or comprises an Fc domain, according to the invention, and/or mediates one or more effector function (e.g. , ADCC, phagocytosis).
  • the diabodies that have been engineered according to the invention are useful for prevention or treatment of cancer, since they have an cytotoxic activity (e.g. , enhanced tumor cell killing and/or enhanced for example, ADCC activity or CDC activity).
  • Cancers associated with a cancer antigen may be treated or prevented by administration of a diabody that binds a cancer antigen and is cytotoxic, and/or has been engineered according to the methods of the invention to exhibit effector function.
  • cancers associated with the following cancer antigens may be treated or prevented by the methods and compositions of the invention: KS 1/4 pan-carcinoma antigen (Perez el al. (1989) " Isolation And Characterization Of A Cdna Encoding The Ksl/4 Epithelial Carcinoma Marker, " J. Immunol. 142:3662-3667; Moller et al. ( 1991) "Bispecific-Monoclonal-Antibody-Directed Lysis Of Ovarian
  • melanoma antigen gp75 (Vijayasardahl et al. (1990) "The Melanoma Antigen Gp75 Is The Human Homologue Of The Mouse B (Brown) Locus Gene Product, " J. Exp. Med.
  • HMW-MAA high molecular weight melanoma antigen
  • melanoma specific antigens such as ganglioside GD2 (Saleh et al. (1993) "Generation Of A Human Antiidiotypic Antibody That Mimics The GD2 Antigen, " J. Immunol., 151 , 3390-3398), ganglioside GD3 (Shitara et al. (1993) "A Mouse/Human Chimeric Anti-(Ganglioside GD3) Antibody With Enhanced Antitumor Activities, " Cancer Immunol. Immunother. 36:373-380), ganglioside GM2 (Livingston et al.
  • tumor-specific transplantation type of cell-surface antigen such as virally-induced tumor antigens including T-antigen DN A tumor viruses and envelope antigens of RNA tumor viruses, oncofetal antigen-alpha-fetoprotein such as CEA of colon, bladder tumor oncofetal antigen (Hellstrom et al. (1985) "Monoclonal Antibodies To Cell Surface Antigens Shared By Chemically Induced Mouse Bladder Carcinomas, " Cancer. Res. 45:2210-2188), differentiation antigen such as human lung carcinoma antigen L6, L20 (Hellstrom et al. (1986) "Monoclonal Mouse Antibodies Raised against Human Lung Carcinoma, " Cancer Res.
  • TSTA tumor-specific transplantation type of cell-surface antigen
  • virally-induced tumor antigens including T-antigen DN A tumor viruses and envelope antigens of RNA tumor viruses
  • oncofetal antigen-alpha-fetoprotein such as CEA of colon
  • the antigen is a T cell receptor derived peptide from a cutaneous T cell lymphoma (see Edelson (1998) "Cutaneous T-Cell Lymphoma: A Model For Selective Immunotherapy, " Cancer J Sci Am. 4:62-71).
  • Cancers and related disorders that can be treated or prevented by methods and compositions of the present invention include, but are not limited to, the following: Leukemias including, but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblasts, promyelocyte, myelomonocytic, monocytic, erythroleukemia leukemias and myelodysplastic syndrome, chronic leukemias such as but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as but not limited to Hodgkin's disease, non-Hodgkin's disease; multiple myelomas such as but not limited to smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia,
  • sarcoma such as but not limited to bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma; brain tumors including but not limited to, glioma, astrocytoma, brain stem glioma, ependymo
  • mucoepidermoid carcinoma, and adenoidcystic carcinoma pharynx cancers including but not limited to, squamous cell cancer, and verrucous
  • skin cancers including but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acral lentiginous melanoma
  • kidney cancers including but not limited to, renal cell cancer, adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell cancer (renal pelvis and/ or uterer); Wilms' tumor
  • bladder cancers including but not limited to, transitional cell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma.
  • cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and papillary adenocarcinomas (for a review of such disorders, see Fishman et al. (1985) Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia; and Murphy et al. (1997) Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books U.S.A., Inc., United States of America).
  • carcinoma including that of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, prostate, cervix, thyroid and skin; including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burketts lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and
  • rhabdomyoscarcoma other tumors, including melanoma, seminoma, tetratocarcinoma, neuroblastoma and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin, including fibrosafcoma, rhabdomyoscarama, and osteosarcoma; and other tumors, including melanoma, xenoderma pegmentosum, keratoactanthoma, seminoma, thyroid follicular cancer and teratocarcinoma.
  • cancers caused by aberrations in apoptosis would also be treated by the methods and compositions of the invention.
  • Such cancers may include but not be limited to follicular lymphomas, carcinomas with p53 mutations, hormone dependent tumors of the breast, prostate and ovary, and precancerous lesions such as familial adenomatous polyposis, and
  • myelodysplastic syndromes In specific embodiments, malignancy or dysproliferative changes (such as metaplasias and dysplasias), or hyperproliferative disorders, are treated or prevented by the methods and compositions of the invention in the ovary, bladder, breast, colon, lung, skin, pancreas, or uterus. In other specific embodiments, sarcoma, melanoma, or leukemia is treated or prevented by the methods and compositions of the invention.
  • a molecule of the invention ⁇ e.g. , a diabody comprising multiple epitope binding domains and, optionally, and Fc domain (or portion thereof)) inhibits or reduces the growth of cancer cells by at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10% relative to the growth of cancer cells in the absence of said molecule of the invention.
  • a molecule of the invention kills cells or inhibits or reduces the growth of cancer cells at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% better than the parent molecule.
  • molecules of the invention comprise an epitope binding domain specific for FcyRIIB and or/ a variant Fc domain (or portion thereof), engineered according to methods of the invention, which Fc domain exhibits greater affinity for FcyRIIB and decreased affinity for FcyRIIIA and/or FcyRIIA relative to a wild-type Fc domain.
  • Molecules of the invention with such binding characteristics are useful in regulating the immune response, e.g., in inhibiting the immune response in connection with autoimmune diseases or inflammatory diseases.
  • FcyRIIB and/or comprising an Fc domain with increased affinity for FcyRIIB and a decreased affinity for FcyRIIIA and/or FcyRIIA may lead to dampening of the activating response to FcyR and inhibition of cellular responsiveness, and thus have therapeutic efficacy for treating and/or preventing an autoimmune disorder.
  • the invention also provides methods for preventing, treating, or managing one or more symptoms associated with an inflammatory disorder in a subject further comprising, administering to said subject a therapeutically or prophylactically effective amount of one or more anti -inflammatory agents.
  • the invention also provides methods for preventing, treating, or managing one or more symptoms associated with an autoimmune disease further comprising, administering to said subject a therapeutically or prophylactically effective amount of one or more immunomodulatory agents.
  • Section 5.7 provides non-limiting examples of anti-inflammatory agents and immunomodulatory agents.
  • alopecia areata ankylosing spondylitis
  • antiphospholipid syndrome autoimmune Addison's disease
  • autoimmune diseases of the adrenal gland autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, cel
  • Hashimoto's thyroiditis idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus erthematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune- mediated diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychrondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man
  • inflammatory disorders include, but are not limited to, asthma, encephilitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic disorders, septic shock, pulmonary fibrosis, undifferentitated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, and chronic inflammation resulting from chronic viral or bacteria infections.
  • COPD chronic obstructive pulmonary disease
  • some autoimmune disorders are associated with an inflammatory condition.
  • an autoimmune disorder there is overlap between what is considered an autoimmune disorder and an inflammatory disorder. Therefore, some autoimmune disorders may also be characterized as inflammatory disorders.
  • inflammatory disorders which can be prevented, treated or managed in accordance with the methods of the invention include, but are not limited to, asthma, encephilitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic disorders, septic shock, pulmonary fibrosis, undifferentitated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, and chronic inflammation resulting from chronic viral or bacteria infections.
  • COPD chronic obstructive pulmonary disease
  • Molecules of the invention comprising at least one epitope binding domain specific for FcyRIIB and/or a variant Fc domain with an enhanced affinity for FcyRIIB and a decreased affinity for FcyRIIIA can also be used to reduce the inflammation experienced by animals, particularly mammals, with inflammatory disorders.
  • a molecule of the invention reduces the inflammation in an animal by at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10% relative to the inflammation in an animal, which is not administered the said molecule.
  • Molecules of the invention comprising at least one epitope binding domain specific for FcyRIIB and/or a variant Fc domain with an enhanced affinity for FcyRIIB and a decreased affinity for FcyRIIIA can also be used to prevent the rejection of transplants.
  • the invention also encompasses methods for treating or preventing an infectious disease in a subject comprising administering a therapeutically or
  • the molecules of the invention are toxic to the infectious agent, enhance immune response against said agent or enhance effector function against said agent, relative to the immune response in the absence of said molecule.
  • Infectious diseases that can be treated or prevented by the molecules of the invention are caused by infectious agents including but not limited to viruses, bacteria, fungi, protozae, and viruses.
  • Viral diseases that can be treated or prevented using the molecules of the invention in conjunction with the methods of the present invention include, but are not limited to, those caused by hepatitis type A, hepatitis type B, hepatitis type C, influenza, varicella, adenovirus, herpes simplex type I (HSV-I), herpes simplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papova virus, cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackie virus, mumps virus, measles virus, rubella virus, polio virus, small pox, Epstein Barr virus, human immunodeficiency virus type I (HIV-I), human immunodeficiency virus type II (HIV-II), and agents of viral diseases such as viral miningitis, encephalitis, dengue or small pox.
  • Bacterial diseases that can be treated or prevented using the molecules of the invention in conjunction with the methods of the present invention, that are caused by bacteria include, but are not limited to, mycobacteria rickettsia, mycoplasma, neisseria, S. pneumonia, Borrelia burgdorferi (Lyme disease), Bacillus antracis (anthrax), tetanus, streptococcus, staphylococcus, mycobacterium, tetanus, pertissus, cholera, plague, diptheria, chlamydia, S. aureus and legionella.
  • Protozoal diseases that can be treated or prevented using the molecules of the invention in conjunction with the methods of the present invention, that are caused by protozoa include, but are not limited to, leishmania, kokzidioa, trypanosoma or malaria.
  • Parasitic diseases that can be treated or prevented using the molecules of the invention in conjunction with the methods of the present invention, that are caused by parasites include, but are not limited to, chlamydia and rickettsia.
  • molecules of the invention comprising at least one epitope binding domain specific for an infectious agent exhibit an antibody effector function towards said agent, e.g. , a pathogenic protein.
  • infectious agents include but are not limited to bacteria (e.g. , Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecials, Candida albicans, Proteus vulgaris, Staphylococcus viridans, and Pseudomonas aeruginosa), a pathogen (e.g., B- lymphotropic papovavirus (LPV); Bordatella pertussis; Borna Disease virus (BDV);
  • LUV B- lymphotropic papovavirus
  • BDV Bordatella pertussis
  • Bovine coronavirus Choriomeningitis virus; Dengue virus; a virus, E. coli; Ebola;
  • Echovirus 1 Echovirus-1 1 (EV); Endotoxin (LPS); Enteric bacteria; Enteric Orphan virus; Enteroviruses ; Feline leukemia virus; Foot and mouth disease virus; Gibbon ape leukemia virus (GALV); Gram-negative bacteria ; Heliobacter pylori; Hepatitis B virus (HBV); Herpes Simplex Virus; HIV-1 ; Human cytomegalovirus; Human coronovirus; Influenza A, B & C ; Legionella; Leishmania mexicana; Listeria monocytogenes; Measles virus; Meningococcus; Morbilliviruses; Mouse hepatitis virus; Murine leukemia virus; Murine gamma herpes virus; Murine retrovirus; Murine coronavirus mouse hepatitis virus; Mycobacterium avium-M; Neisseria gonorrhoeae; Newcastle disease virus; Parvovirus B 19; Plasmodium falciparum; Po

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US20130295121A1 (en) 2013-11-07
MX339622B (es) 2016-06-02
AU2011286024B2 (en) 2014-08-07
MX2013001102A (es) 2013-05-09
IL224515A (en) 2016-07-31
CA2807127A1 (en) 2012-02-09
JP2013540696A (ja) 2013-11-07
ES2667100T3 (es) 2018-05-09
CN103154025B (zh) 2015-07-01
BR112013002533A2 (pt) 2021-09-08
US9889197B2 (en) 2018-02-13
AU2011286024A1 (en) 2013-02-21
CA2807127C (en) 2019-02-12
SG187682A1 (en) 2013-03-28
JP5964300B2 (ja) 2016-08-03
EP2601216A4 (de) 2013-12-25
WO2012018687A1 (en) 2012-02-09
CN103154025A (zh) 2013-06-12
EP2601216B1 (de) 2018-01-03

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